WO2000043543A1 - Detection of differences between polynucleotides - Google Patents

Abstract

A method is disclosed for detecting the presence of a difference between two related nucleic acid sequences. A complex is formed comprising both of the nucleic acid sequences in double stranded form. The complex comprises at least one pair of non-complementary strands and each of the non-complementary strands within the complex has a label. The complex is subjected to conditions wherein, if a difference between the two related nucleic acid sequences is present, strand exchange in the complex ceases and wherein, if no difference between the two related nucleic acid sequences is present, strand exchange in the complex continues until complete strand exchange occurs. A first signal is detected from the association of the labels as part of the complex. The association of the labels is related to the presence of the difference. A complex is also formed comprising the nucleic acid sequence suspected of comprising a difference in double stranded form and a predetermined amount of a non-relevant reference polynucleotide in double stranded form. The complex comprises at least one pair of non-complementary strands and each of the non-complementary strands within the complex has a label. A second signal from the association of the labels as part of the above complex is detected. A ratio of the first signal to the second signal is determined and related to the presence of the difference.

Description

DETECTION OF DIFFERENCES BETWEEN POLYNUCLEOTIDES

BACKGROUND OF THE INVENTION 1 Field of the Invention

Nucleic acid hybridization has been employed for investigating the identity and establishing the presence of nucleic acids Hybridization is based on complementary base pairing When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules The ability of single stranded deoxyπbonucleic acid (ssDNA) or πbonucleic acid (RNA) to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research The availability of radioactive nucleoside tπphosphates of high specific activity and the 32p labeling of DNA with T4 polynucleotide kinase has made it possible to identify, isolate, and characterize various nucleic acid sequences of biological interest

Nucleic acid hybridization has great potential in diagnosing disease states associated with unique nucleic acid sequences These unique nucleic acid sequences may result from genetic or environmental change in DNA by insertions, deletions, point mutations, or by acquiring foreign DNA or RNA by means of infection by bacteria molds, fungi and viruses Nucleic acid hybridization has, until now, been employed primarily in academic and industrial molecular biology laboratories The application of nucleic acid hybridization as a diagnostic tool in clinical medicine is limited because of the frequently very low concentrations of disease related DNA or RNA present in a patient's body fluid and the unavailability of a sufficiently sensitive method of nucleic acid hybridization analysis

One method for detecting specific nucleic acid sequences generally involves immobilization of the target nucleic acid on a solid support such as nitrocellulose paper, cellulose paper, diazotized paper, or a nylon membrane After the target nucleic acid is fixed on the support, the support is contacted with a suitably labeled probe nucleic acid for about two to forty-eight hours After the above time period, the solid support is washed several times at a controlled temperature to remove unhybπdized probe The support is then dried and the hybridized material is detected by autoradiography or by spectrometπc methods

When very low concentrations must be detected, the above method is slow and labor intensive and nonisotopic labels that are less readily detected than radiolabels are frequently not suitable

A method for the enzymatic amplification of specific segments of DNA known as the polymerase chain reaction (PCR) method has been described This in vitro amplification procedure is based on repeated cycles of denaturation, oligonucleotide primer annealing and primer extension by thermophilic polymerase, resulting in the exponential increase in copies of the region flanked by the primers The PCR primers which anneal to opposite strands of the DNA, are positioned so that the polymerase catalyzed extension product of one primer can serve as a template strand for the other, leading to the accumulation of a discrete fragment whose length is defined by the distance between the hybridization sites on the DNA sequence complementary to the 5' ends of the oligonucleotide primers Other methods for amplifying nucleic acids are single primer amplification, ligase chain reaction (LCR), nucleic acid sequence based amplification (NASBA) and the Q-beta-replicase method Regardless of the amplification used, the amplified product must be detected Genetic recombination involves the exchange of DNA strands between two related DNA duplexes The branch point between two duplex DNA's that have exchanged a pair of strands is thought to be an important intermediate in homologous recombination This branch point is otherwise referred to as the Holliday junction Movement of the Holliday junction by branch migration can increase or decrease the amount of genetic information exchanged between homologues In vitro strand exchange is protein mediated, unlike the spontaneous migration that occurs in vitro

There is a great demand for simple universal high-throughput methods for detection of differences in related nucleic acid sequences regardless of the exact nature of the difference This demand is becoming more and more urgent due to the ongoing rapid discovery of new disease related mutations brought about by the progress of the Human Genome Project A detection method for mutations that is not dependent on the exact location of the mutation is valuable in the case of diseases that are known to result from various mutations within a given sequence. Moreover, such a method will be useful for verification of sequence homology as related to various applications in molecular biology, molecular medicine and population genetics. Current methods are either targeted for sets of known mutations, such as, for example the Reverse Dot Blot method, or involve gel- based techniques, such as, for example, single stranded conformational polymorphism (SSCP) denaturing gradient gel electrophoresis (DGGE) or direct sequencing as well as a number of methods for the detection of heteroduplexes. Accordingly, such methods are laborious and time consuming.

It is desirable to have a sensitive, simple, inexpensive method for detecting differences in nucleic acids such as mutations, preferably, in a homogeneous format. The method should minimize the number and complexity of steps and reagents and provide a normalized result Such a method would be suitable for a large-scale population screening

2. Description of the Related Art U.S. Patent Application Serial No. 08/771 ,623 (Lishanski. et al.) describes the detection of differences in nucleic acids

Formation of a single base mismatch that impedes spontaneous DNA branch migration is described by Panyutin, eial , (1993) J. Mol. Biol.. 230:413-424.

The kinetics of spontaneous DNA branch migration is discussed by Panyutin, et al , (1994) Proc Natl Acad Sci. USA, 91_.: 2021-2025.

European Patent Application No 0 450 370 A1 (Wetmur et al.,) discloses branch migration of polynucleotides

A displacement polynucleotide assay method and polynucleotide complex reagent therefor is discussed in U S Patent No 4, 766,062 (Diamond, et al.,). A strand displacement assay and complex useful therefor is discussed in PCT application WO 94/06937 (Eadie, et al.,).

PCT application WO/86/06412 (Fritsch, et al.,) discusses process and nucleic acid construct for producing reagent complexes useful in determining target nucleotide sequences.

A process for amplifying, detecting and/or cloning nucleic acid sequences is disclosed in U.S. Patent Nos. 4,683,195.

SUMMARY OF THE INVENTION One method in accordance with the present invention is directed to detecting the presence of a difference between two related nucleic acid sequences. A complex is formed comprising both of the nucleic acid sequences in double stranded form. The complex comprises at least one pair of non- complementary strands and each of the non-complementary strands within the complex has a label. The complex is subjected to conditions wherein, if a difference between the two related nucleic acid sequences is present, strand exchange in the complex ceases and wherein, if no difference between the two related nucleic acid sequences is present, strand exchange in the complex continues until complete strand exchange occurs. A first signal is detected from the association of the labels as part of the complex. The association of the labels is related to the presence of the difference. A complex is also formed comprising the nucleic acid sequence suspected of comprising a difference in double stranded form and a predetermined amount of a non-relevant reference polynucleotide in double stranded form. The complex comprises at least one pair of non- complementary strands and each of the non-complementary strands within the complex has a label. A second signal from the association of the labels as part of the above complex is detected. A ratio of the first signal to the second signal is determined and related to the presence of the difference.

Another aspect of the present invention is a method for detecting a mutation within a target nucleic acid sequence. A tailed target partial duplex A' is formed from the target sequence and comprises a duplex of two nucleic acid strands of the target sequence, a label and at one end of the duplex, two non-complementary oligonucleotides, one linked to each of the strands A combination is provided comprising the tailed target partial duplex A' and a tailed first reference partial duplex B' lacking the mutation and having a label as a part thereof The tailed reference partial duplex B' is comprised of two nucleic acid strands Each of the strands is complementary, respectively, to a strand in the tailed target partial duplex A but for the possible presence of a mutation The labels are present in non-complementary strands of the tailed target and tailed reference partial duplexes, respectively The combination is subjected to conditions wherein, if a mutation is present strand exchange in the complex ceases and wherein, if no mutation is present, strand exchange in the complex continues until complete strand exchange occurs A first signal is detected by means of the labels resulting from the formation of a complex between the tailed partial duplexes, the formation thereof being directly related to the presence of the mutation A combination is provided comprising the tailed target partial duplex A' and a tailed second reference partial duplex B' having a label as a part thereof The tailed second reference partial duplex B' is comprised of two nucleic acid strands of a non- relevant polynucleotide wherein the labels are present in non-complementary strands of the tailed target and tailed second reference partial duplexes, respectively The combination is subjected to conditions wherein a complex comprising a four-stranded structure is formed A second signal is detected by means of the labels resulting from the formation of the above complex A ratio of the first signal to the second signal is determined and related to the presence of the mutation

Another aspect of the present invention is a method of detecting a mutation within a target nucleic acid sequence The target sequence is amplified by polymerase chain reaction, using primers P1 and P2 to produce an amplicon AA One of the primers P1 and P2 comprises a label The primer P1 is comprised of a 3'-end portion Pa that can hybridize with the target sequence and 5'-end portion B1 that cannot hybridize with the target sequence A primer P3 is extended by chain extension along one strand of amplicon AA to produce a tailed target partial duplex A' Primer P3 is comprised of the 3'-end portion Pa and a 5'-end portion A1 that cannot hybridize to the target sequence or its complement A first reference nucleic acid sequence is amplified using the primer P2 and the primer P3, by polymerase chain reaction to produce amplicon BB The first reference sequence is identical to the target sequence but lacks a possible mutation Primer P2 comprises a label when the primer P2 above comprises a label and the primer P3 comprises a label when the primer P1 above comprises a label Primer P1 is extended by chain extension along one strand of amplicon BB to produce a tailed reference partial duplex B'. The tailed target partial duplex A' is allowed to bind to the tailed first reference partial duplex B' A first signal is detected from the labels as a result of the formation of a complex between the tailed partial duplexes, the formation thereof being directly related to the presence of the mutation. A second reference nucleic acid sequence is amplified, using a set of primers, by polymerase chain reaction to produce amplicon CC The reference sequence is non-relevant to the target sequence The primers comprise priming sequences for the second reference and respectively, the 5'-end portion B1 and the 5'-end portion A1 wherein one of the primers comprises a label. Primer P1 is extended by chain extension along one strand of amplicon CC to produce a tailed reference partial duplex C. The tailed target partial duplex A' is allowed to bind to the tailed reference partial duplex C A second signal is detected from the labels as a result of the formation of a complex comprising a four-stranded structure between the tailed partial duplexes above A ratio of the first signal to the second signal is determined and related to the presence of the mutation.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1A is a schematic diagram of an embodiment wherein a mutation is present in the target nucleic acid sequence and branch migration is stopped. Fig B is a schematic diagram showing complete strand exchange in the absence of a mutation in the target nucleic acid sequence, there being no difference between target and reference nucleic acid sequences

Fig 2A is a schematic diagram of an embodiment wherein a mutation is present in the target nucleic acid sequence and branch migration is stopped

Fig 2B is a schematic diagram showing complete strand exchange in the absence of a mutation in the target nucleic acid sequence, there being no difference between target and reference nucleic acid sequences

Fig 3 is a schematic diagram of an embodiment wherein a mutation is present in the target nucleic acid sequence and branch migration is stopped

Fig 4 is a schematic diagram of an embodiment in accordance with the present invention wherein a mutation is present in the target nucleic acid sequence and a non-relevant nucleic acid sequence is employed in a branch migration, which is stopped because of the differences between the target nucleic acid sequence and the non-relevant nucleic acid sequence

Fig 5 is a depiction of the M tuberculosis (tb) rpoB gene sequence

DESCRIPTION OF THE SPECIFIC EMBODIMENTS The present invention provides an improvement in the method of Lishanski, et al The method is universal and permits detection of any difference in two related nucleic acid sequences whether or not such difference is known Such differences include any mutation including single base substitution, deletion or insertion within a sequence that can be defined by a pair of primers for conducting the polymerase chain reaction The method may be homogeneous or heterogeneous and non-radioactive The present method is fast, provides a normalized result and is amenable to automation It is ideally suited for rapid mutation pre-screenmg The invention also has application in the area of amplification by polymerase chain reaction The present invention permits PCR and subsequent steps such as detection of the PCR products, to be conducted without the need for additional probes in a single container without a separation step

The present method involves formation of a four-strand DNA structure or complex from DNA The formation involves producing two partial duplexes by amplification by using three different primers in the polymerase chain reaction and allowing the amplified products to anneal The complex dissociates into normal duplex structures by strand exchange by means of branch migration when the hybridized portions of each partial duplex are identical However, where there is a difference between the two hybridized portions, the complex does not dissociate and can be detected as an indication of the presence of a difference between the nucleic acids A particularly attractive feature of the present invention is that the reactions may be carried out simultaneously in the same medium without a separation step

We have designed a method for normalizing the signal obtained in the present method The normalization method is based on formation of stable four stranded polynucleotide structures when amplification product from test polynucleotide is mixed with similarly produced products of amplification of a non- relevant reference sequence

Before proceeding further with a description of the specific embodiments of the present invention a number of terms will be defined

Nucleic acid --a compound or composition that is a polymeric nucleotide or polynucleotide The nucleic acids include both nucleic acids and fragments thereof from any source in purified or unpuπfied form including DNA (dsDNA and ssDNA) and RNA, including t-RNA, m-RNA, r-RNA, mitochondnal DNA and RNA, chloroplast DNA and RNA, DNA-RNA hybrids, or mixtures thereof, genes, chromosomes, plasmids, the genomes of biological material such as microorganisms, e g bacteria, yeasts, viruses, viroids, molds, fungi, plants, animals, humans, and the like The nucleic acid can be only a minor fraction of a complex mixture such as a biological sample The nucleic acid can be obtained from a biological sample by procedures well known in the art Also included are genes, such as hemoglobin gene for sickle-cell anemia cystic fibrosis gene, oncogenes cDNA and the like Where the nucleic acid is RNA it is first converted to cDNA by means of a primer and reverse transcriptase The nucleotide polymerase used in the present invention for carrying out amplification and chain extension can have reverse transcriptase activity Sequences of interest may be embedded in sequences of any length of the chromosome, cDNA, plasmid, etc

Sample -- the material suspected of containing the nucleic acid Such samples include biological fluids such as blood, serum, plasma, sputum, lymphatic fluid, semen vaginal mucus, feces urine, spinal fluid and the like, biological tissue such as hair and skin and so forth Other samples include cell cultures and the like, plants food forensic samples such as paper fabrics and scrapings, water, sewage, medicinals, etc When necessary, the sample may be pretreated with reagents to liquefy the sample and release the nucleic acids from binding substances Such pretreatments are well known in the art

Amplification of nucleic acids -- any method that results in the formation of one or more copies of a nucleic acid (exponential amplification) One such method for enzymatic amplification of specific sequences of DNA is known as the polymerase chain reaction (PCR) as described by Saiki, et al , supra This in vitro amplification procedure is based on repeated cycles of denaturation, oligonucleotide primer annealing and primer extension by thermophilic template dependent polynucleotide polymerase, resulting in the exponential increase in copies of the desired sequence of the nucleic acid flanked by the primers The two different PCR primers are designed to anneal to opposite strands of the DNA at positions that allow the polymerase catalyzed extension product of one primer to serve as a template strand for the other, leading to the accumulation of a discrete double stranded fragment whose length is defined by the distance between the 5' ends of the oligonucleotide primers Primer length can vary from about 10 to 50 or more nucleotides and are usually selected to be at least about 15 nucleotides to ensure high specificity The double stranded fragment that is produced is called an "amplicon and may vary in length form as few as about 30 nucleotides to 10,000 or more

Chain extension of nucleic acids -- extension of the 3'-end of a polynucleotide in which additional nucleotides or bases are appended Chain extension relevant to the present invention is template dependent that is, the appended nucleotides are determined by the sequence of a template nucleic acid to which the extending chain is hybridized The chain extension product sequence that is produced is complementary to the template sequence Usually, chain extension is enzyme catalyzed preferably, in the present invention, by a thermophilic DNA polymerase

Target nucleic acid sequence - a sequence of nucleotides to be studied either for the presence of a difference from a related sequence or for the determination of its presence or absence The target nucleic acid sequence may be double stranded or single stranded When the target nucleic acid sequence is single stranded the method of the present invention produces a nucleic acid duplex comprising the single stranded target nucleic acid sequence

The target sequence usually exists within a portion or all of a nucleic acid, the identity of which is known to an extent sufficient to allow preparation of various primers necessary for introducing one or more priming sites flanking the target sequence or conducting an amplification of the target sequence or a chain extension of the products of such amplification in accordance with the present invention Accordingly other than for the sites to which the primers bind, the identity of the target nucleic acid sequence may or may not be known In general, in PCR, primers hybridize to, and are extended along (chain extended), at least the target sequence, and thus, the target sequence acts as a template The target sequence usually contains from about 30 to 20,000 or more nucleotides, more frequently, 100 to 10 000 nucleotides, preferably, 50 to 1 ,000 nucleotides The target nucleic acid sequence is generally a fraction of a larger molecule or it may be substantially the entire molecule The minimum number of nucleotides in the target sequence is selected to assure that a determination of a difference between two related nucleic acid sequences in accordance with the present invention can be achieved

Reference nucleic acid sequence -- a nucleic acid sequence that is related to the target nucleic acid in that the two sequences are identical except for the presence of a difference, such as a mutation Where a mutation is to be detected, the reference nucleic acid sequence usually contains the normal or "wild type" sequence. In certain situations the reference nucleic acid sequence may be part of the sample as, for example, in samples from tumors, the identification of partially mutated microorganisms, or identification of heterozygous carriers of a mutation. Consequently, both the reference and the target nucleic acid sequences are subjected to similar or the same amplification conditions As with the target nucleic acid sequence, the identity of the reference nucleic acid sequence need be known only to an extent sufficient to allow preparation of various primers necessary for introducing one or more priming sites flanking the reference sequence or conducting an amplification of the target sequence or a chain extension of the products of such amplification in accordance with the present invention. Accordingly, other than for the sites to which the primers bind, the identity of the reference nucleic acid sequence may or may not be known. The reference nucleic acid sequence may be a reagent employed in the methods in accordance with the present invention This is particularly the situation where the present method is used in PCR amplification for detection of a target nucleic acid sequence. Depending on the method of preparation of this reagent it may or may not be necessary to know the identity of the reference nucleic acid. The reference nucleic acid reagent may be obtained from a natural source or prepared by known methods such as those described below in the definition of oligonucleotides.

Non-relevant reference nucleic acid sequence - a sequence of nucleotides that differs from the reference nucleic acid sequence, e.g , wild type sequence, in that there is no correspondence between the non-relevant sequence and the target nucleic acid. In other words, although there may be some common nucleotides between the non-relevant sequence and the target nucleic acid, there is not sufficient correspondence such that the non-relevant sequence has the same or similar hybridization patterns as the target nucleic acid The non-relevant reference nucleic acid reagent may be obtained from a natural source or prepared by known methods such as those described below in the definition of oligonucleotides The non-relevant reference nucleic acid sequence may be a nucleic acid sequence from another organism or a different gene sequence for the same organism

Holliday junction -- the branch point in a four way junction in a complex of two identical nucleic acid sequences and their complementary sequences The junction is capable of undergoing branch migration resulting in dissociation into two double stranded sequences where sequence identity and complementarity extend to the ends of the strands

Complex -- a complex of four nucleic acid strands containing a Holliday junction, which is inhibited from dissociation into two double stranded sequences because of a difference in the sequences and their complements Accordingly, the complex is quadramolecular

Related nucleic acid sequences ~ two nucleic acid sequences are related when they contain at least 15 nucleotides at each end that are identical but have different lengths or have intervening sequences that differ by at least one nucleotide Frequently related nucleic acid sequences differ from each other by a single nucleotide Such difference is referred to herein as the "difference between two related nucleic acid sequences " A difference can be produced by the substitution, deletion or insertion of any single nucleotide or a series of nucleotides within a sequence

Mutation - a change in the sequence of nucleotides of a normally conserved nucleic acid sequence resulting in the formation of a mutant as differentiated from the normal (unaltered) or wild type sequence Mutations can generally be divided into two general classes namely base-pair substitutions and frameshift mutations The latter entail the insertion or deletion of one to several nucleotide pairs A difference of one nucleotide can be significant as to phenotypic normality or abnormality as in the case of, for example, sickle cell anemia Partial duplex - a fully complementary double stranded nucleic acid sequence wherein one end thereof has non-complementary oligonucleotide sequences, one linked to each strand of the double stranded molecule, each non- complementary sequence having 8 to 60, preferably, 10 to 50, more preferably, 15 to 40, nucleotides. Thus, the partial duplex is said to be "tailed" because each strand of the duplex has a single stranded oligonucleotide chain linked thereto.

Duplex — a double stranded nucleic acid sequence wherein all of the nucleotides therein are complementary.

Oligonucleotide - a single stranded polynucleotide, usually a synthetic polynucleotide. The oligonucleotide(s) are usually comprised of a sequence of 10 to 100 nucleotides, preferably, 20 to 80 nucleotides, and more preferably, 30 to 60 nucleotides in length.

Various techniques can be employed for preparing an oligonucleotide utilized in the present invention. Such oligonucleotide can be obtained by biological synthesis or by chemical synthesis. For short sequences (up to about 100 nucleotides) chemical synthesis will frequently be more economical as compared to the biological synthesis. In addition to economy, chemical synthesis provides a convenient way of incorporating low molecular weight compounds and/or modified bases during the synthesis step. Furthermore, chemical synthesis is very flexible in the choice of length and region of the target polynucleotide binding sequence. The oligonucleotide can be synthesized by standard methods such as those used in commercial automated nucleic acid synthesizers. Chemical synthesis of DNA on a suitably modified glass or resin can result in DNA covalently attached to the surface. This may offer advantages in washing and sample handling. For longer sequences standard replication methods employed in molecular biology can be used such as the use of M13 for single stranded DNA as described by J. Messing (1983) Methods Enzvmol, 101 , 20-78.

Other methods of oligonucleotide synthesis include phosphotriester and phosphodiester methods (Narang, ET aL (1979) Meth. Enzvmol 68: 90) and synthesis on a support (Beaucage, et a], (1981 ) Tetrahedron Letters 22: 1859- 1862) as well as phosphoramidate technique Caruthers, M H et al_, "Methods in Enzymology," Vol 154 pp 287-314 (1988) and others described in "Synthesis and Applications of DNA and RNA " S A Narang editor, Academic Press, New York, 1987, and the references contained therein Oligonucleotide prιmer(s) -- an oligonucleotide that is usually employed in a chain extension on a polynucleotide template such as in, for example, an amplification of a nucleic acid The oligonucleotide primer is usually a synthetic oligonucleotide that is single stranded, containing a hybπdizable sequence at its 3'-end that is capable of hybridizing with a defined sequence of the target or reference polynucleotide Normally the hybπdizable sequence of the oligonucleotide primer has at least 90%, preferably 95%, most preferably 100%, complementarity to a defined sequence or primer binding site The number of nucleotides in the hybπdizable sequence of an oligonucleotide primer should be such that stringency conditions used to hybridize the oligonucleotide primer will prevent excessive random non-specific hybridization Usually, the number of nucleotides in the hybπdizable sequence of the oligonucleotide primer will be at least ten nucleotides, preferably at least 15 nucleotides and, preferably 20 to 50, nucleotides In addition the primer may have a sequence at its 5'-end that does not hybridize to the target or reference polynucleotiΦes that can have 1 to 60 nucleotides, preferably 8 to 30 polynucleotides

Nucleoside tπphosphates - nucleosides having a 5'-tπphosphate substituent The nucleosides are pentose sugar derivatives of nitrogenous bases of either puπne or pyrimidine derivation, covalently bonded to the 1 '-carbon of the pentose sugar which is usually a deoxyπbose or a πbose The puπne bases comprise adenιne(A) guanine (G) inosine (I), and derivatives and analogs thereof The pyrimidine bases comprise cytosine (C), thymine (T), uracil (U), and derivatives and analogs thereof Nucleoside tπphosphates include deoxyπbonucleoside tπphosphates such as the four common tπphosphates dATP, dCTP, dGTP and dTTP and πbonucleoside tπphosphates such as the four common tπphosphates rATP, rCTP, rGTP and rUTP The term "nucleoside tπphosphates" also includes derivatives and analogs thereof, which are exemplified by those derivatives that are recognized and polymerized in a similar manner to the undeπvatized nucleoside tπphosphates Examples of such derivatives or analogs, by way of illustration and not limitation, are those which are biotinylated amine modified, alkylated, and the like and also include phosphorothioate, phosphite, ring atom modified derivatives, and the like Nucleotide - a base-sugar-phosphate combination that is the monomeπc unit of nucleic acid polymers, i e DNA and RNA

Modified nucleotide - is the unit in a nucleic acid polymer that results from the incorporation of a modified nucleoside tπphosphate during an amplification reaction and therefore becomes part of the nucleic acid polymer

Nucleoside -- is a base-sugar combination or a nucleotide lacking a phosphate moiety

Nucleotide polymerase -- a catalyst, usually an enzyme, for forming an extension of a polynucleotide along a DNA or RNA template where the extension is complementary thereto The nucleotide polymerase is a template dependent polynucleotide polymerase and utilizes nucleoside tπphosphates as building blocks for extending the 3'-end of a polynucleotide to provide a sequence complementary with the polynucleotide template Usually, the catalysts are enzymes, such as DNA polymerases, for example, prokaryotic DNA polymerase (I, II, or III), T4 DNA polymerase T7 DNA polymerase, Klenow fragment, and reverse transcriptase and are preferably thermally stable DNA polymerases such as Vent DNA polymerase, VentR DNA polymerase, Pfu DNA polymerase, Taq DNA polymerase, and the like, derived from any source such as cells, bacteria, such as E. coli. plants, animals, virus, thermophilic bacteria, and so forth

Wholly or partially sequentially - when the sample and various agents utilized in the present invention are combined other than concomitantly (simultaneously), one or more may be combined with one or more of the remaining agents to form a subcombination Subcombination and remaining agents can then be combined and can be subjected to the present method Hybridization (hybridizing) and binding-in the context of nucleotide sequences these terms are used interchangeably herein The ability of two nucleotide sequences to hybridize with each other is based on the degree of complementarity of the two nucleotide sequences, which in turn is based on the fraction of matched complementary nucleotide pairs The more nucleotides in a given sequence that are complementary to another sequence, the more stringent the conditions can be for hybridization and the more specific will be the binding of the two sequences Increased stringency is achieved by elevating the temperature, increasing the ratio of cosolvents, lowering the salt concentration,

Complementary-Two sequences are complementary when the sequence of one can bind to the sequence of the other in an anti-parallel sense wherein the 3' -end of each sequence binds to the 5'-end of the other sequence and each A, T(U), G, and C of one sequence is then aligned with a T(U), A, C, and G, re- spectively, of the other sequence

Copy - means a sequence that is a direct identical copy of a single stranded polynucleotide sequence as differentiated from a sequence that is complementary to the sequence of such single stranded polynucleotide

Conditions for extending a primer -- includes a nucleotide polymerase, nucleoside triphosphates or analogs thereof capable of acting as substrates for the polymerase and other materials and conditions required for enzyme activity such as a divalent metal ion (usually magnesium), pH, ionic strength, organic solvent (such as formamide) and the like

Member of a specific binding pair ("sbp member")~one of two different molecules, having an area on the surface or in a cavity which specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of the other molecule The members of the specific binding pair are referred to as gand and receptor (anti gand) These may be members of an immunological pair such as antigen-antibody, or may be operator-repressor, nuclease-nucleotide biotin-avidm hormone-hormone receptor, IgG-protein A, DNA-DNA, DNA-RNA and the like

Ligand-any compound for which a receptor naturally exists or can be prepared Receptor ("antιlιgand")-any compound or composition capable of recognizing a particular spatial and polar organization of a molecule, e g , epitopic or determinant site Illustrative receptors include naturally occurring and synthetic receptors, e g thyroxine binding globulin, antibodies, enzymes, Fab fragments, lectins, nucleic acids repressors oligonucleotides, protein A, complement component C1 q or DNA binding proteins and the like

Small organic molecule-a compound of molecular weight less than about 1500, preferably 100 to 1000, more preferably 300 to 600 such as biotin, digoxin, fluorescem, rhodamine and other dyes, tetracycline and other protein binding molecules, and haptens, etc The small organic molecule can provide a means for attachment of a nucleotide sequence to a label or to a support

Support or surface-a porous or non-porous water insoluble material The support can be hydrophi c or capable of being rendered hydrophilic and includes inorganic powders such as silica magnesium sulfate, and alumina, natural polymeric materials, particularly cellulosic materials and materials derived from cellulose, such as fiber containing papers, e g , filter paper, chromatographic paper, etc , synthetic or modified naturally occurring polymers, such as nitrocellulose cellulose acetate poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose, polyacrylate polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate poly(ethylene terephthalate), nylon, poly(vιnyl butyrate), etc , either used by themselves or in conjunction with other materials, glass available as Bioglass, ceramics, metals, and the like Natural or synthetic assemblies such as liposomes phospho pid vesicles, and cells can also be employed

Binding of sbp members to a support or surface may be accomplished by well-known techniques commonly available in the literature See, for example, "Immobilized Enzymes." Ichiro Chibata, Halsted Press, New York (1978) and Cuatrecasas, J. Biol. Chem., 245:3059 (1970). The surface can have any one of a number of shapes, such as strip_: rod, particle, including bead, and the like.

Label - a member of a signal producing system. Labels include reporter molecules that can be detected directly by virtue of generating a signal, and specific binding pair members that may be detected indirectly by subsequent binding to a cognate that contains a reporter molecule such as oligonucleotide sequences that can serve to bind a complementary sequence or a specific DNA binding protein; organic molecules such as biotin or digoxigenin that can bind respectively to streptavidin and antidigoxin antibodies, respectively; polypeptides; polysaccharides; and the like. In general, any reporter molecule that is detectable can be used. The reporter molecule can be isotopic or nonisotopic, usually non- isotopic, and can be a catalyst, such as an enzyme, dye, fluorescent molecule, chemiluminescer, coenzyme, enzyme substrate, radioactive group, a particle such as latex or carbon particle, metal sol, crystallite, liposome, cell, etc., which may or may not be further labeled with a dye, catalyst or other detectable group, and the like. The reporter group can be a fluorescent group such as fluorescein, a chemiluminescent group such as luminol, a terbium chelator such as N- (hydroxyethyl) ethylenediaminetriacetic acid that is capable of detection by delayed fluorescence, and the like.

The label is a member of a signal producing system and can generate a detectable signal either alone or together with other members of the signal producing system. As mentioned above, a reporter molecule can serve as a label and can be bound directly to a nucleotide sequence. Alternatively, the reporter molecule can bind to a nucleotide sequence by being bound to an sbp member complementary to an sbp member that comprises a label bound to a nucleotide sequence. Examples of particular labels or reporter molecules and their detection can be found in U.S. Patent Application Serial No. 07/555,323 filed July 19, 1990, the relevant disclosure of which is incorporated herein by reference. Signal Producing System— the signal producing system may have one or more components, at least one component being the label The signal producing system generates a signal that relates to the presence of a difference between the target polynucleotide sequence and the reference polynucleotide sequence The signal producing system includes all of the reagents required to produce a measurable signal When a reporter molecule is not conjugated to a nucleotide sequence, the reporter molecule is normally bound to an sbp member complementary to an sbp member that is bound to or part of a nucleotide sequence Other components of the signal producing system can include substrates, enhancers, activators, chemilummescent compounds cofactors, inhibitors, scavengers, metal ions, specific binding substances required for binding of signal generating substances, coenzymes, substances that react with enzymic products, enzymes and catalysts, and the like The signal producing system provides a signal detectable by external means, such as by use of electromagnetic radiation, electrochemical detection, desirably by spectrophotometπc detection The signal-producing system is described more fully in U S Patent Application Serial No 07/555,323, filed July 19, 1990, the relevant disclosure of which is incorporated herein by reference

Ancillary Materials-Various ancillary materials will frequently be employed in the methods and assays carried out in accordance with the present invention For example, buffers will normally be present in the assay medium, as well as stabilizers for the assay medium and the assay components Frequently, in addition to these additives, proteins may be included, such as albumins, organic solvents such as formamide, quaternary ammonium salts, polycations such as dextran sulfate, surfactants, particularly non-ionic surfactants, binding enhancers, e g , polyalkylene glycols, or the like

As mentioned above, one aspect of the present invention concerns a method for detecting the presence of a difference between two related nucleic acid sequences In the method, if there is a difference between the two related nucleic acid sequences a stable quadramolecular complex is formed comprising both of the nucleic acid sequences in double stranded form Usually, the complex comprises a Holliday junction Both members of at least one pair of non- complementary strands within the complex have labels A first signal is obtained from the association of the labels as part of the complex as an indication of the presence of the difference between the two related sequences A complex is also formed comprising the nucleic acid sequence suspected of comprising a difference in double stranded form and a predetermined amount of a non-relevant reference polynucleotide in double stranded form The complex comprises at least one pair of non-complementary strands and each of the non-complementary strands within the complex has a label A second signal from the association of the labels as part of the above complex is detected A ratio of the first signal to the second signal is determined and related to the presence of the difference

The method may be employed for detecting the presence of a mutation in a target nucleic acid sequence or for detecting the presence of a target nucleic acid sequence. A discussion of the underlying reactions that form the present method follows. Referring to Fig 1A, quadramolecular complex C comprises partial duplex A and partial duplex B Partial duplexes A' and B' are related in that their hybridized portions are identical except for mutation M in partial duplex A Additionally, partial duplex A' has a label L1 , which may or may not differ from label L2 in partial duplex B' Oligonucleotide tail A1 of partial duplex A' is hybridized to corresponding oligonucleotide tail B2 of partial duplex B' and, similarly, oligonucleotide tail A2 of partial duplex A' is hybridized to oligonucleotide tail B1 of partial duplex B' Accordingly complex C is quadramolecular and contains a four way junction H Because oligonucleotide tails A1 and B1 are different, branch migration can only proceed away from these tails and then only until mutation M is reached, at which point branch migration stops Thus, when a mutation is present, complex C is stable and can be detected by determining whether both labels L1 and L2 have become associated The association of the labels indicates the presence of complex C and thus the presence of mutation M in the target nucleic acid sequence If mutation M is not present (see Fig 1 B), branch migration continues until complete strand exchange occurs and only separate duplexes D and E are present whereupon no complex C is detected.

Fig. 2A concerns a mutation within a target nucleic acid sequence A that contains mutation M A tailed target partial duplex A' is formed from the target sequence and is comprised of a duplex of the target sequence, a label L1 and at one end of the duplex, two non-complementary oligonucleotides A1 and A2, one linked to each strand of duplex A' Oligonucleotides A1 and A2 have from 8 to 60 nucleotides, preferably, 15 to 30 nucleotides The tailed target partial duplex is provided in combination with a labeled tailed reference partial duplex B' lacking mutation M. The tailed reference partial duplex B' is comprised of two nucleic acid strands that are complementary to the strands in A' but for mutation M Accordingly, one terminus of the tailed reference partial duplex B' has, as the end part of each strand, a sequence of nucleotides B1 and B2, respectively, that are complementary to A2 and A1 , respectively, of A' and are not complementary to each other. Labels L1 and L2 are present in non-complementary strands of the tailed target and tailed reference partial duplexes A' and B', respectively, where L1 and L2 may be the same or different

A complex C is formed as described above for Fig. 1A. Oligonucleotide tail A1 of A is hybridized to corresponding oligonucleotide tail B2 of B' and, similarly, oligonucleotide tail A2 of A' is hybridized to oligonucleotide tail B1 of B'. Because oligonucleotide tails A1 and B1 are different, branch migration can only proceed away form these tails and then only until mutation M is reached, at which point branch migration stops Thus, when a mutation is present, complex C is stable and can be detected by determining whether both labels L1 and L2 have become associated. The association of the labels indicates the presence of complex C. The formation of complex C is directly related to the presence of the mutation. If mutation M is not present in the nucleic acid (see Fig 2B), branch migration continues until complete strand exchange has occurred and only the separate duplexes D and E are present In this event no complex C is detected. Fig 3 depicts by way of example and not limitation the production of tailed target partial duplex A' from target nucleic acid duplex A having mutation M and the production of tailed reference partial duplex B from reference nucleic acid duplex B In Fig 3 A is amplified by the polymerase chain reaction (PCR) using primers P1 and P2 to produce an amplicon AA The amplification may be carried out separately from that of the reference nucleic acid sequence or in the presence of the reference nucleic acid sequence For the normalization method discussed hereinbelow, the amplification of the target nucleic acid sequence should be carried out separately from that of the reference nucleic acid sequence Primer P2 contains a label L1 and primer P1 is comprised of a 3'-end portion Pa that can hybridize with the target sequence and 5 -end portion B1 that cannot hybridize with the target sequence The amplification is carried out in the presence of a nucleotide polymerase and nucleoside triphosphates using temperature cycling Amplicon AA has two strands, a labeled strand derived from primer P2 and an unlabeled strand derived from primer P1 The unlabeled strand has a 5'-end portion B1 of primer P1 and the labeled strand has a corresponding 3'-end portion A2, which is the complement of B1

The above amplification is carried out by PCR utilizing temperature cycling to achieve denaturation of duplexes, oligonucleotide primer annealing, and primer extension by thermophilic template dependent nucleotide polymerase In conducting PCR amplification of nucleic acids, the medium is cycled between two to three temperatures The temperatures for the present method for the amplification by PCR generally range from about 50°C to about 100°C, more usually, from about 50°C to about 95°C Relatively low temperatures of from about 50°C to about 80°C are employed for the hybridization steps, while denaturation is carried out at a temperature of from about 80°C to about 100°C and extension is carried out at a temperature of from about 70°C to about 80°C, usually about 72°C to about 74°C The amplification is conducted for a time sufficient to achieve a desired number of copies for an accurate determination of whether or not two related nucleic acids have a difference Generally, the time period for conducting the method is from about 10 seconds to about 10 minutes per cycle and any number of cycles can be used from 1 to as high as about 60 or more, usually about 10 to about 50, frequently, about 20 to about 45 As a matter of convenience it is usually desirable to minimize the time period and the number of cycles In general the time period for a given degree of amplification can be minimized, for example by selecting concentrations of nucleoside tripnosphates sufficient to saturate the polynucleotide polymerase, by increasing the concentrations of polynucleotide polymerase and polynucleotide primer and by using a reaction container that provides for rapid thermal equilibration Generally the time period for conducting the amplification in the method of the invention is from about 5 to about 200 minutes

In an example of a typical temperature cycling as may be employed, the medium is subjected to multiple temperature cycles of heating at about 90°C to about 100°C for about 10 seconds to about 3 minutes and cooling to about 70°C to about 80°C for a period of about 10 seconds to about 3 minutes Referring again to Fig 3 a chain extension of primer P3 along the labeled strand of amplicon AA occurs to produce tailed target partial duplex A' Primer P3 is comprised of a 3'-end portion Pa which is identical to Pa of primer P1 and which binds to the labeled strand of AA P3 has 5'-end portion A1 that is not complementary to amplicon AA The chain extension occurs in the presence of a nucleotide polymerase and nucleoside triphosphates under appropriate temperature conditions to produce the complementary strand of the labeled strand Although copies thereof may be produced, these are not shown in Fig 3 for purposes of simplicity It has been found that such copies do not interfere with the branch migration procedures described herein To avoid production of such copies primers P2 and P1 may be removed prior to extension of P3 in a manner as described hereinbelow The complementary unlabeled strand of tailed target partial duplex A has a 5'-end portion A1 , which is not complementary to the 3'-end portion A2 of the labeled strand of A' Unless the PCR reaction is carried out to produce an excess of the labeled strand, there will also be present the unlabeled strand from the amplification This strand is not a template during chain extension to form partial duplex A'

The conditions for carrying out the chain extension in accordance with the present invention are similar to those for the amplification described above Preferably, the medium is subjected to heating at about 90°C to about 100°C for a period of about 10 seconds to about 3 minutes, cooling to about 50°C to about 65°C for a period of about 10 seconds to about 2 minutes and heating to about 70°C to about 80°C for a period of about 30 seconds to about 5 minutes

Referring to Fig 3, for the normalization method of the present invention, reference nucleic acid sequence B is amplified by PCR preferably in a separate medium Primer P2 and primer P3 are employed in a polymerase chain reaction to produce amplicon BB The amplification is carried out using temperature cycling under the conditions described above in the presence of a nucleotide polymerase and nucleoside triphosphates B is comprised of a sequence identical to A except for mutation M Generally, primer P2 used for this amplification contains a label L2 that may be the same as or different than L1 Amplicon BB has two strands, a labeled strand derived from primer P2 and an unlabeled strand derived from primer P3 The unlabeled strand has end portion A1 of primer P3 and the labeled strand has corresponding end portion B2, which is the complement of A1 A chain extension of primer P1 along the labeled strand of amplicon BB is carried out, under the conditions mentioned above for the chain extension of primer P3 along the labeled strand in duplex AA, to produce tailed reference partial duplex B' As mentioned above primer P1 is comprised of portion Pa, which binds to the labeled strand of BB and portion B1 that does not bind to amplicon BB The chain extension is carried out in the presence of a nucleotide polymerase and nucleoside triphosphates under appropriate temperature conditions so that only the complement of the labeled strand is produced and not a copy The extended primer P1 has a 5'-end portion B1 , which is not complementary to end portion B2 of the labeled strand of B' As can be seen, A' and B' are related in that each of their labeled strands is complementary except for mutation M to the unlabeled strand of the other

The reaction mixtures above for the PCR amplification reactions for target nucleic acid sequence A and reference nucleic acid sequence B are combined where the above amplifications were carried out separately The combined reaction mixtures are subjected to conditions for branch migration The strands of partial duplexes A' and B' are allowed to bind and undergo branch migration by combining the mixtures containing partial duplexes A' and B' and incubating the combination at a temperature of about 30°C to about 75°C, preferably about 60°C to about 70°C, for at least about one minute, preferably, about 20 to about 60 minutes, wherein complex C is formed as described above for Figs 1 and 2 Oligonucleotide tail A1 of A is hybridized to corresponding oligonucleotide tail B2 of B' and, similarly, oligonucleotide tail A2 of A' is hybridized to oligonucleotide tail B1 of B' Branch migration within complex C continues under the above temperature conditions with separation of the complex into duplexes D and E unless a mutation M is present, whereupon branch migration and strand dissociation is inhibited Complex C is then detected the presence of which is directly related to the presence of mutation M

Referring to Fig 3, labels L1 and L2 are incorporated into the partial duplexes that comprise complex C and provide a means for detection of complex C This is by way of illustration and not limitation and other convenient methods for detecting complex C may be employed, such as the use of a receptor for the complex In this approach there is required only one label, L1 or L2, which comprises an sbp member or a reporter molecule A receptor for the sbp member and a receptor that can bind to complex C by virtue of a feature other than L1 or L2 can both bind to complex C and provide a means for detection

As mentioned above, for the present invention the above reactions are carried out independently to produce tailed partial duplexes A' and B', respectively, in separate reaction mixtures Then, the reaction mixtures can be combined to allow the respective strands of A and B' to bind to one another to form complex C If a normalization method in accordance with the present invention was not to be carried out, the above reactions can be carried out in the same reaction medium and many or all of the reactions preferably are carried out simultaneously. In this approach a combination is provided in a single medium. The combination comprises (i) a sample containing a target nucleic acid sequence suspected of having a mutation, (ii) a reference nucleic acid sequence, which may be added separately if it is not known to be present in the sample and which corresponds to the target nucleic acid lacking the mutation, which as explained above may be the wild type nucleic acid, (iii) a nucleotide polymerase, (iv) nucleoside triphosphates, and (v) primers P1 , P2 and P3, wherein P2 may include primer P2 labeled with L1 and primer P2 labeled with L2, or P2 may be unlabeled and primers P1 and P3 may be labeled respectively with L1 and L2. The medium is then subjected to multiple temperature cycles of heating and cooling to simultaneously achieve all of the amplification and chain extension reactions described above with reference to Fig. 3 except that in this embodiment there is no need to avoid making copies of any of the extended primers. Preferably, in this embodiment, each cycle includes heating the medium at about 90^CC to about 100°C for about 10 seconds to about 3 minutes, cooling the medium to about 60°C to about 70°C for a period of about 10 seconds to about 3 minutes, and heating the medium at about 70°C to about 75°C for a period of about 10 seconds to about 3 minutes although different temperatures may be required depending on the lengths of the primer sequences. Following the above temperature cycling the medium is subjected to heating for a period of time sufficient to denature double stranded molecules, preferably, at about 90°C to about 99°C for about 10 seconds to about 2 minutes, and cooled to about 40°C to about 80°C, preferably about 60°C to about 70°C, and held at this temperature for at least about one minute, preferably for about 20 minutes to about 1 hours.

Following cooling of the medium all possible partial and complete duplexes are formed that can form from 1 ) single strands that have any combination of reference or mutant sequences and 5'-ends A2 and B2, and 2) single strands having any combination of reference or mutant sequences and 5'-ends A1 or B1 wherein the strands may further be labeled with either L1 or L2 when L1 and L2 are different Among the partial duplexes that are formed are the tailed partial duplexes A' and B', which can bind to each other to form complex C, which does not dissociate into duplexes D and E when a mutation is present A determination of the presence of such a complex is then made to establish the presence of a mutation in the target nucleic acid sequence When primers P1 and P3 are labeled instead of primer P2, the labels L1 and L2 in partial duplexes A and B' are attached to tails A1 and B1 , respectively, which still provides for detection of complex C when a mutation is present

While all the steps of this determination are preferably carried out in the same medium as that used for the above reactions, some or all of the steps can be carried out wholly or partially sequentially in different media Thus, for example, PCR amplification of target sequence A and target sequence B, each using primers P1 , P2 and P3, can be conducted in separate solutions The solution can then be combined, heated to about 90°C to about 100°C to denature strands and then incubated as before at about 40°C to about 80°C to permit formation of duplexes and complex C when a mutation is present Detection of complex C can then be carried out directly in the combined solutions or by adding reagents required for detection or by separating the complex C, for example, on a solid surface, and detecting its presence on the surface

As mentioned above, we have designed a method for normalizing the signal obtained in the above method The normalization method is based on formation of stable four stranded polynucleotide structures when amplification product from target polynucleotide is mixed with similarly produced products of amplification of a non-relevant reference sequence As explained above, such a sequence differs from the above reference sequence, e g , wild type sequence in that there is no correspondence between the non-relevant sequence and the test polynucleotide Thus, any non-relevant polynucleotide may be used in the normalization procedure. An important feature of this normalization method is that the non- relevant reference sequence be treated to incorporate oligonucleotide tails that are the same tails as used for the target polynucleotide Accordingly, the primers used in the PCR amplification of the non-relevant reference sequence comprise the same tails as the tails in the primers employed in the PCR amplification of the target polynucleotide Of course the primers for the PCR amplification of the non- relevant reference sequence also comprise appropriate priming sequences that are capable of hybridizing to the non-relevant reference sequence in accordance with standard PCR methodology The length of the primers for the non-relevant reference sequence is determined employing considerations that are similar to those for the relevant reference sequence as discussed above

In carrying out the normalization procedure, two signals are obtained The first signal is obtained as described above, with reference to Fig 3, for the target polynucleotide and the relevant reference sequence or wild type sequence Ahquots of the PCR amplification products from the target polynucleotide and the relevant reference sequence are combined and subjected to branch migration conditions A signal, i e , the first signal, is obtained The signal is representative of both the specific difference such as genotype, of the target polynucleotide and the amount of the target polynucleotide

Similarly, an aliquot from the PCR amplification of the target polynucleotide is combined with an aliquot that contains the PCR amplification product of a predetermined amount of the non-relevant reference sequence The combination is subjected to branch migration conditions as discussed above for the target polynucleotide and the relevant reference The PCR amplification products from the target polynucleotide and the non-relevant reference sequence both contain the same oligonucleotide tails Thus, during the branch migration conditions, there is cross-hybridization of the tails between the two products leading to four-stranded structures between the two products Because of the difference in the sequences of the target polynucleotide and the non-relevant reference sequence, these four- stranded structures are stable and do not dissociate In so far as the two sequences are not related, signals are obtained from all target polynucleotides regardless of the specific genotype The signal obtained, namely the second signal, is indicative of only the amount of the target polynucleotide since the amount of the non-relevant reference sequence is predetermined An evaluation of the ratio of the first signal to the second signal is made and is related to the presence of the mutation in the test polynucleotide independent of its initial concentration In other words, the ratio of the signals provides normalized signals and represents target polynucleotide genotype regardless of the amount of the target polynucleotide in the sample under evaluation

The above normalization method in accordance with the present invention is next described with reference to Fig 4 As can be seen the PCR amplification of target nucleic acid sequence A is shown on the left The product of the PCR amplification is A' The PCR amplification of the non-relevant reference nucleic acid sequence E is shown on the right in Fig 4 The product of this PCR amplification is E' As discussed above, these PCR amplification reactions are carried out separately The reaction mixture containing the PCR amplification product of target nucleic acid sequence A is an aliquot of the reaction mixture discussed above with reference to Fig 3 An aliquot of this reaction mixture containing A' is combined with an aliquot of the PCR amplification reaction mixture of non-relevant reference nucleic acid sequence E, which is present in a predetermined amount By the term "predetermined amount" is meant that a known amount of the non-relevant reference E is employed in order that the ratio of the signals in accordance with the normalization procedure of the present invention provide for a sensitive and accurate determination Usually, this predetermined amount is the same as that predicted for the target sequence, or up to 10 fold more than or up to 10 fold less than, that predicted for the target sequence

Referring to Fig 4, non-relevant reference nucleic acid sequence E is amplified by PCR in a separate medium Primer P4 and primer P5 are employed in a polymerase chain reaction to produce amplicon EE The amplification is carried out using temperature cycling under the conditions described above in the presence of a nucleotide polymerase and nucleoside triphosphates E is comprised of a non-relevant sequence and thus the primers employed correspond to the non-relevant sequence Generally, primer P4 used for this amplification contains a label L2 that may be the same as or different than L1 Amplicon EE has two strands, a labeled strand derived from primer P4 and an unlabeled strand derived from primer P5 The unlabeled strand has end portion A1 of primer P3 and the labeled strand has corresponding end portion B2, which is the complement of A1

A chain extension of primer P6 along the labeled strand of amplicon EE is carried out, under the conditions mentioned above for the chain extension of primer P3 along the labeled strand in duplex AA, to produce tailed non-relevant reference partial duplex E' As mentioned above, primer P6 is comprised of portion Pb, which binds to the labeled strand of EE and portion B1 that does not bind to amplicon EE The chain extension is carried out in the presence of a nucleotide polymerase and nucleoside triphosphates under appropriate temperature conditions so that only the complement of the labeled strand is produced and not a copy The extended primer P6 has a 5'-end portion B1 , which is not complementary to end portion B2 of the labeled strand of E' As can be seen, it is important A and E' be unrelated but that tails A1 and B1 be the same for primers the set or primers used for amplification of target nucleic acid sequence A and non-relevant nucleic acid sequence E

In carrying out the present method, an aqueous medium is employed Other polar cosolvents may also be employed, usually oxygenated organic solvents of from 1-6, more usually from 1 -4 carbon atoms, including alcohols, ethers and the like Usually these cosolvents if used, are present in less than about 70 weight percent, more usually in less than about 30 weight percent

The pH for the medium is usually in the range of about 4 5 to 9 5, more usually in the range of about 5 5 - 8 5, and preferably in the range of about 6 - 8, usually about 8 In general for amplification, the pH and temperature are chosen and varied, as the case may be so as to cause, either simultaneously or sequentially, dissociation of any internally hybridized sequences, hybridization of the oligonucleotide primer with the target nucleic acid sequence, extension of the primer, and dissociation of the extended primer. Various buffers may be used to achieve the desired pH and maintain the pH during the determination. Illustrative buffers include borate, phosphate, carbonate, Tris, barbital and the like._. The particular buffer employed is not critical to this invention but in individual methods one buffer may be preferred over another. The buffer employed in the present methods normally contains magnesium ion (Mg²⁺), which is commonly used with many known polymerases, although other metal ions such as manganese have also been used. Preferably, magnesium ion is used at a concentration of from about 1 to about 20mM, preferably, from about 4 to about 10mM. The magnesium can be provided as a salt, for example, magnesium chloride and the like. The primary consideration is that the metal ion permit the distinction between different nucleic acids in accordance with the present invention. The concentration of the nucleotide polymerase is usually determined empirically. Preferably, a concentration is used that is sufficient such that further increase in the concentration does not decrease the time for the amplification by over 5-fold, preferably 2-fold. The primary limiting factor generally is the cost of the reagent. The amount of the target nucleic acid sequences that is to be examined in accordance with the present invention can be as low as one or two molecules in a sample. The priming specificity of the primers used for the detection of a difference between two related nucleic acids and other factors will be considered with regard to the need to conduct an initial amplification of the target nucleic acid. It is within the purview of the present invention for detection of a mutation to carry out a preliminary amplification reaction to increase, by a factor of 10² or more, the number of molecules of the target nucleic acid sequence. The amplification can be by any convenient method such as PCR, amplification by single primer, NASBA, and so forth, but will preferably be by PCR. The amount of the target nucleic acid sequence to be subjected to subsequent amplification using primers in accordance with the present invention may vary from about 1 to about 101 °, more usually from about 103 to about 108 molecules, preferably at least about 10-21 M in the medium and may be about 10- 10 to about 10-19M, _more usually about 10-14 to about 10-19M

If an initial amplification of the target nucleic acid sequence is carried out to increase the number of molecules it may be desirable, but not necessary, to remove, destroy or inactivate the primers used in the initial amplification depending on the nature of the protocol utilized Accordingly, when the present method is carried out using step-wise addition of reagents for each separate reaction, such as, for example, in the embodiment of Fig 3, primer P1 should be removed prior to the extension of primer P3 On the other hand, for example, in the embodiment described hereinbelow where the reactions are carried out simultaneously, it is not necessary to remove any of the primers An example, by way of illustration and not limitation, of an approach to destroy the primers is to employ an enzyme that can digest only single stranded DNA For example, an enzyme may be employed that has both 5' to 3' and 3' to 5 exonuclease activities, such as, e g , exo VII The medium is incubated at a temperature and for a period of time sufficient to digest the primers Usually incubation at about 20°C to about 40°C for a period of about 10 to about 60 minutes is sufficient for an enzyme having the above activity The medium is next treated to inactivate the enzyme, which can be accomplished, for example, by heating for a period of time sufficient to achieve inactivation Inactivation of the enzyme can be realized usually upon heating the medium at about 90°C to about 100°C for about 0 5 to about 30 minutes Other methods of removing the primers will be suggested to those skilled in the art It has been found, however, that removal of such primers is not necessary in carrying out the methods of the invention

The amount of the oligonucleotide pπmer(s) used in the amplification reaction in the present invention will be at least as great as the number of copies desired and will usually be about 10-9 to about 10-3 M, preferably, about 10-7 to about 10-4 M Preferably, the concentration of the oligonucleotide pπmer(s) is substantially in excess over, preferably at least about 100 times greater than, more preferably, at least 1000 times greater than, the concentration of the target nucleic acid sequence The concentration of the nucleoside triphosphates in the medium can vary widely, preferably, these reagents are present in an excess amount for both amplification and chain extension The nucleoside triphosphates are usually present in about 10-6 to about 10-2M, preferably about 10-5 to about 10-3M

The order of combining the various reagents may vary The target nucleic acid may be combined with a pre-prepared combination of primers unlabeled P2, labeled P2, and P1 , nucleoside triphosphates and nucleotide polymerase

Alternatively the target nucleic acid for example, can be combined with only unlabeled primer P2 together with the nucleoside triphosphates and polymerase After temperature cycling is carried out, the reaction mixture can be combined with the remaining primers P1 and labeled P2 As mentioned above, the identity of the target nucleic acid sequence does not need to be known except to the extent to allow preparation of the necessary primers for carrying out the above reactions The present invention permits the determination of the presence or absence of a mutation in a nucleic acid in a sample without the need to fully identify the sequence of the nucleic acid Accordingly, one is able to determine the presence of a mutation in a nucleic acid between two sequences of nucleotides for which primers can be made

In the present invention one means of detecting the quadramolecular complex involves the use of two labels on non-complementary strands The labels become associated by virtue of both being present in the quadramolecular complex if a difference is present between the related sequences Detection of the two labels in the complex provides for detection of the complex Generally, the association of the labels within the complex is detected This association may be detected in many ways For example, one of the labels can be an sbp member and a complementary sbp member is provided attached to a support Upon the binding of the complementary sbp members to one another, the complex becomes bound to the support and is separated from the reaction medium The other label employed is a reporter molecule that is then detected on the support The presence of the reporter molecule on the support indicates the presence of the complex on the support, which in turn indicates the presence of the mutation in the target nucleic acid sequence. An example of a system as described above is the enzyme-linked immunosorbent assay (ELISA), a description of which is found in "Enzyme-lmmunoassay," Edward T Maggio, editor, CRC Press, Inc., Boca Raton, Florida (1980) wherein, for example the sbp member is biotin, the complementary sbp member is streptavidin and the reporter molecule is an enzyme such as alkaline phosphatase

Detection of the signal will depend upon the nature of the signal producing system utilized. If the reporter molecule is an enzyme, additional members of the signal producing system would include enzyme substrates and so forth. The product of the enzyme reaction is preferably a luminescent product, or a fluorescent or non-fluorescent dye, any of which can be detected spectrophotometrically, or a product that can be detected by other spectrometric or electrometric means. If the reporter molecule is a fluorescent molecule, the medium can be irradiated and the fluorescence determined Where the label is a radioactive group, the medium can be counted to determine the radioactive count. The association of the labels within the complex may also be determined by using labels that provide a signal only if the labels become part of the complex. This approach is particularly attractive when it is desired to conduct the present invention in a homogeneous manner Such systems include enzyme channeling immunoassay, fluorescence energy transfer immunoassay, electrochemiluminescence assay, induced luminescence assay, latex agglutination and the like.

In one aspect of the present invention detection of the complex is accomplished by employing at least one suspendable particle as a support, which may be bound directly to a nucleic acid strand or may be bound to an sbp member that is complementary to an sbp member attached to a nucleic acid strand. Such a particle serves as a means of segregating the bound target polynucleotide sequence from the bulk solution for example by settling, electrophoretic separation or magnetic separation A second label, which becomes part of the complex if a mutation is present is a part of the signal producing system that is separated or concentrated in a small region of the solution to facilitate detection Typical labels that may be used in this particular embodiment are fluorescent labels, particles containing a sensitizer and a chemiluminescent olefin (see U S Serial No 07/923,069 filed July 31 , 1992 the disclosure of which is incorporated herein by reference), chemiluminescent and electroluminescent labels Preferably, the particle itself can serve as part of a signal producing system that can function without separation or segregation The second label is also part of the signal producing system and can produce a signal in concert with the particle to provide a homogeneous assay detection method A variety of combinations of labels can be used for this purpose When all the reagents are added at the beginning of the reaction, the labels are limited to those that are stable to the elevated temperatures used for amplification, chain extension, and branch migration In that regard it is desirable to employ as labels polynucleotide or polynucleotide analogs having 5 to 20 or more nucleotides depending on the nucleotides used and the nature of the analog Polynucleotide analogs include structures such as polyπbonucleotides, polynucleoside phosphonates, peptido-nucleic acids, polynucleoside phosphorothioates homo DNA and the like In general, unchanged nucleic acid analogs provide stronger binding and shorter sequences can be used Included in the reaction medium are oligonucleotide or polynucleotide analogs that have sequences of nucleotides that are complemen- tary One of these oligonucleotides is attached to, for example, a reporter molecule or a particle The other is attached to a primer, either primer P2 or primer P1 and/or P3 as a label Neither the oligonucleotide nor polynucleotide analog should serve as a polynucleotide polymerase template This is achieved by using either a polynucleotide analog or a polynucleotide that is connected to the primer by an abasic group The abasic group comprises a chain of 1 to 20 or more atoms, preferably at least 2 atoms, more preferably, 6 to 12 atoms such as, for example, carbon, hydrogen, nitrogen, oxygen sulfur, and phosphorus, which may be present as various groups such as polymethylenes, polymethylene ethers, hydroxylated polymethylenes, and so forth The abasic group conveniently may be introduced into the primer during solid phase synthesis by standard methods

Under the proper annealing temperature an oligonucleotide or polynucleotide analog attached to a reporter molecule or particle can bind to its complementary polynucleotide analog or oligonucleotide separated by an abasic site that has become incorporated into partial duplexes A' and B' as labels during amplification If the partial duplexes become part of a quadramolecular complex, the reporter molecule or particle becomes part of the complex By using different polynucleotide analogs or oligonucleotide sequences for labels, L1 and L2, two different reporter molecules or particles can become part of the complex Various combinations of particles and reporter molecules can be used The particles, for example, may be simple latex particles or may be particles comprising a sensitizer chemiluminescer, fluorescer, dye, and the like Typical particle/reporter molecule pairs include a dye crystallite and a fluorescent label where binding causes fluorescence quenching or a tπtiated reporter molecule and a particle containing a scintillator Typical reporter molecule pairs include a fluorescent energy donor and a fluorescent acceptor dye Typical particle pairs include (1) two latex particles, the association of which is detected by light scattering or turbidimetry, (2) one particle capable of absorbing light and a second label particle which fluoresces upon accepting energy from the first, and (3) one particle incorporating a sensitizer and a second particle incorporating a chemiluminescer as described for the induced luminescence immunoassay referred to in U S. Serial No 07/704,569, filed May 22, 1991 , entitled "Assay Method Utilizing Induced Luminescence", which disclosure is incorporated herein by reference

Briefly, detection of the quadramolecular complex using the induced luminescence assay as applied in the present invention involves employing a photosensitizer as part of one label and a chemiluminescent compound as part of the other label If the complex is present the photosensitizer and the chemiluminescent compound come into close proximity The photosensitizer generates singlet oxygen and activates the chemiluminescent compound when the two labels are in close proximity The activated chemiluminescent compound subsequently produces light The amount of light produced is related to the amount of the complex formed

By way of illustration as applied to the present invention, a particle is employed, which comprises the chemiluminescent compound associated therewith such as by incorporation therein or attachment thereto The particles have a recognition sequence usually an oligonucleotide or polynucleotide analog, attached thereto with a complementary sequence incorporated into one of the nucleic acid strands as a label, L1 Another particle is employed that has the photosensitizer associated therewith These particles have a recognition sequence attached thereto, which is different than that attached to the chemiluminescent particles A complementary sequence is incorporated as a label L2 in the nucleic acid strand in complex C that is not complementary to the nucleic acid strand carrying label L1 Once the medium has been treated in accordance with the present invention to form a quadramolecular complex C, the medium is irradiated with light to excite the photosensitizer, which is capable in its excited state of activating oxygen to a singlet state Because the chemiluminescent compound of one of the sets of particles is now in close proximity to the photosensitizer by virtue of the presence of the target polynucleotide having a mutation, the chemiluminescent compound is activated by the singlet oxygen and emits luminescence The medium is then examined for the presence and/or the amount of luminescence or light emitted the presence thereof being related to the presence of quadramolecular complex C The presence of the latter indicates the presence and/or amount of the target polynucleotide having a mutation or of the target polynucleotide itself As a matter of convenience predetermined amounts of reagents employed in the present invention can be provided in a kit in packaged combination A kit can comprise in packaged combination (a) a pπmer P2 that is extendable along one of the strands of the target and reference nucleic acid sequences, (b) a primer P1 comprising a 3'-end portion Pa that binds to and is extendable along the other of the strands of the target and reference nucleic acid sequences and a 5'-end portion B1 that does not bind to the target and reference nucleic acid sequences, and (c) a primer P3 comprising 3'-end portion Pa and a portion A1 that is different from B1 and does not bind to the target and reference nucleic acid sequences Preferably, primer P2 can be labeled but primers P1 and P3 alternatively may be labeled The kit further contains reagents for carrying out a normalization procedure as described above Such reagents comprise a set of primers for the amplification of a non-relevant reference sequence as well as the non-relevant sequence itself The kit can also include a reference nucleic acid, which corresponds to a target nucleic acid sequence except for the possible presence of a difference such as a mutation, and reagents for conducting an amplification of target nucleic acid sequence prior to subjecting the target nucleic acid sequence to the methods of the present invention The kit can also include nucleoside triphosphates and a nucleotide polymerase The kit can further include particles as described above capable of binding to the label on at least one of the primers The kit can further include members of a signal producing system and also various buffered media, some of which may contain one or more of the above reagents Preferably, primers P1 , P2 and P3 are packaged in a single container and P4, P5 and P6 are packaged in a single container

The relative amounts of the various reagents in the kits can be varied widely to provide for concentrations of the reagents that substantially optimize the reactions that need to occur during the present method and to further substantially optimize the sensitivity of the method in detecting a mutation Under appropriate circumstances one or more of the reagents in the kit can be provided as a dry pow- der, usually lyophilized including excipients which on dissolution will provide for a reagent solution having the appropriate concentrations for performing a method or assay in accordance with the present invention Each reagent can be packaged in separate containers or some or all of the reagents can be combined in one con- tamer where cross-reactivity and shelf life permit The kits may also include a written description of a method in accordance with the present invention as described above

EXAMPLES The invention is demonstrated further by the following illustrative examples

Temperatures are in degrees centigrade (°C) and parts and percentages are by weight, unless otherwise indicated The following definitions and abbreviations are used herein

Tris - Tπs(hydroxymethyl)amιnomethane-HCI (a 10X solution) from BioWhittaker, Walkersville, MD

BSA - bovine serum albumin from Gibco BRL, Gaithersburg MD bp - base pairs wt (+) - wild type allele mut (-) - mutant allele Target sample - DNA sample to be tested for the presence of a mutation,

Reference sample - DNA sample homozygous for the wt sequence with which target samples are challenged sec - seconds hr - hours mm - minutes

Buffer A - 10mM Tπs-HCI (pH8 3) 50mM KCI, 1 5mM MgCI₂ 200 μg/ml BSA

Buffer B - 100mM Tπs-HCI (pH8 3), 500mM KCI, 15mM MgCI₂ 2 mg/ml BSA Buffer C - 0 1 M Tris 0 3M NaCl 25 mM EDTA, 0 1 % BSA, 0 1 % dextran T- 500, a 1 320 dilution of mouse IgG (HBR-1 from Scantibodies Laboratory Inc , Los Angeles, CA), 0 05% Kathon (Rohm and Haas, Philadelphia, PA), and 0 01 % gentamycm sulfate RLU - relative light units nt - nucleotides

MAD - maleimidylaminodextran

Ab - antibody

Sav - streptavidin MOPS - 3-(N-morpholιno)propane sulfonic acid sulfo-SMCC - sulfosuccinimidyl 4-(N-maleιmιdomethyl)cyclohexane-1 - carboxylate

NHS - N-hydroxysuccinimide

EDAC - 1 -ethyl-3-(3-dιmethylamιnopropyl)carbodιιmιde hydrochlonde DMSO - dimethylsulfoxide

MES - morphohnoethanesulfonate rpm - rotations per m

EDTA - ethylenediaminetetraacetic acid

SATA - N-succinimidyl S-acetylthioacetate BSA - bovine serum albumin from Sigma Chemical Company, St Louis MO eq - equivalents bp - base pairs

A₂₈₀ - absorbance at wavelength 280 nanometers

DPP - 4,7-dιphenylphenanthrolιne Eu(TTA)₃ - europium tπ-3-(2-thιenoyl)-1 ,1 ,1 -tπfluoroacetonate

L or I - liter exo VII - exonuclease VII from E coli (from Amersham Life Science) (USB).

DMF - dimethyl formamide

THF - tetrahydrofuran MS - mass spectroscopy NMR - nuclear magnetic resonance spectroscopy TMSCl - tetramethylsilylchloπde

ELISA - enzyme linked immunosorbent assay as described in "Enzyme- Immunoassay Edward T Maggio CRC Press, Inc , Boca Raton Florida (1980) Monoclonal antibodies were produced by standard hybrid cell technology

Briefly, the appropriated immunogen was injected into a host, usually a mouse or other suitable animal, and after a suitable period of time the spleen cells from the host were obtained Alternatively, unsensitized cells from the host were isolated and directly sensitized with the immunogen in vitro Hybrid cells were formed by fusing the above cells with an appropriate myeloma cell line and cultuπng the fused cells The antibodies produced by the cultured hybrid cells were screened for their binding affinity to the particular antigen, dig-BSA conjugate A number of screening techniques were employed such as, for example, ELISA screens Selected fusions were then recloned Beads

Acc-AbDig - Acceptor beads coupled (MAD) to the anti-Dig antibody (with 377 antibody molecules per bead) were prepared as follows

Hydroxypropylaminodextran (1 NH₂/ 7 glucose) was prepared by dissolving Dextran T-500 (Pharmacia, Uppsala Sweden) (50g) in 150 mL of H₂O in a 3-neck round-bottom flask equipped with mechanical stirrer and dropping funnel To the above solution was added 18 8g of Zn (BF₄)₂ and the temperature was brought to 87°C with a hot water bath Epichlorohydπn (350mL) was added dropwise with stirring over about 30 mm while the temperature was maintained at 87-88°C The mixture was stirred for 4 hr while the temperature was maintained between 80°C and 95°C, then the mixture was cooled to room temperature Chlorodextran product was precipitated by pouring slowly into 3L of methanol with vigorous stirring, recovered by filtration and dried overnight in a vacuum oven

The chlorodextran product was dissolved in 200mL of water and added to 2L of concentrated aqueous ammonia (36%) This solution was stirred for 4 days at room temperature, then concentrated to about 190mL on a rotary evaporator The concentrate was divided into two equal batches, and each batch was precipitated by pouring slowly into 2L of rapidly stirring methanol. The final product was recovered by filtration and dried under vacuum.

Hydroxypropylaminodextran (1 NH₂/ 7 glucose), prepared above, was dissolved in 50mM MOPS, pH 7.2, at 12.5 mg/mL. The solution was stirred for 8 hr at room temperature, stored under refrigeration and centrifuged for 45 min at 15,000 rpm in a Sorvall RC-5B centrifuge immediately before use to remove a trace of solid material. To 10mL of this solution was added 23.1 mg of Sulfo-SMCC in 1 mL of water. This mixture was incubated for 1 hr at room temperature and used without further purification.

C-28 thioxene was prepared as follows: To a solution of 4-bromoaniline (30g, 174mmol) in dry DMF (200mL) was added 1- bromotetradecane (89.3mL, 366mmol) and N,N-diisopropylethylamine (62.2mL, 357mmol). The reaction solution was heated at 90°C for 16 hr under argon before being cooled to room temperature. To this reaction solution was again added 1- bromotetradecane (45mL, 184mmol) and N,N-diisopropylethylamine (31 mL, 178mmol) and the reaction mixture was heated at 90°C for another 15 hr. After cooling, the reaction solution was concentrated in vacuo and the residue was diluted with CH₂CI₂ (400mL). The CH₂CI₂ solution was washed with 1 N aqueous NaOH (2x), H₂0. and brine, was dried over Na₂SO₄ and was concentrated in vacuo to yield a dark brown oil (about 110g). Preparative column chromatography on silica gel by a Waters 500 Prep LC system eluting with hexane afforded a yellow oil that contained mainly the product (4-bromo-N,N-di-(Cι₄H₂₉)-aniline) along with a minor component 1-bromotetradecane. The latter compound was removed from the mixture by vacuum distillation (boiling point 105-110°C, 0.6mm) to leave 50.2g (51%) of the product as a brown oil. To a mixture of magnesium turnings (9.60g, 395mmol) in dry THF (30mL) under argon was added dropwise a solution of the above substituted aniline product (44.7g, 79mmol) in THF (250mL). A few crystals of iodine were added to initiate the formation of the Grignard reagent. When the reaction mixture became warm and began to reflux, the addition rate was regulated to maintain a gentle reflux After addition was complete the mixture was heated at reflux for an additional hour The cooled supernatant solution was transferred via cannula to an addition funnel and added dropwise (over 2 5 hr) to a solution of phenylglyoxal (1 1 7g 87mmol) in THF (300mL) at -30°C under argon The reaction mixture was gradually warmed to 0°C over 1 hr and stirred for another 30 mm The resulting mixture was poured into a mixture of ice water (800mL) and ethyl acetate (250mL) The organic phase was separated and the aqueous phase was extracted with ethyl acetate (3x) The combined organic phases were washed with H₂0 (2x), brine and were dried over MgS0₄ Evaporation of the solvent gave 48 8g of the crude product as a dark green oily liquid Flash column chromatography of this liquid (gradient elution with hexane 1 5 98 5 3 97, 5 95 ethyl acetate hexane) afforded 24 7g (50%) of the benzoin product (MS (C₄₂H₆₉NO₂) [M-H]⁺ 618 6 ¹H NMR (250 MHz, CDCI₃) was consistent with the expected benzoin product To a solution of the benzoin product from above (24.7g, 40mmol) in dry toluene (500mL) was added sequentially 2- mercaptoethanol (25g 320mmol) and TMSCl (100mL, 788mmol) The reaction solution was heated at reflux for 23 hr under argon before being cooled to room temperature To this was added additional TMSCl (50mL, 394mmol), and the reaction solution was heated at reflux for another 3 hr The resulting solution was cooled, was made basic with cold 2 5N aqueous NaOH and was extracted with CH₂CI₂ (3x) The combined organic layers were washed with saturated aqueous NaHCO₃ (2x) and brine was dried over Na₂SO₄ and was concentrated in vacuo to give a brown oily liquid Preparative column chromatography on silica gel by using a Waters 500 Prep LC system (gradient elution with hexane, 1 99, 2 98 ethyl acetate-hexane) provided 15 5g (60%) of the C-28 thioxene as an orange-yellow oil (MS (C₄₄H₇₁NOS) [M-Hf 661 6 ¹H NMR (250 MHz, CDCI₃) was consistent with the expected C-28 thioxene product 2-(4-(N,N-dι-(C ₄H₂₉)-anιlιno)-3-phenyl thioxene

Carboxyl chemiluminescer (acceptor) beads (TAR beads) The following dye composition was employed 20% C-28 thioxene (prepared as described above), 1 6% 1 -chloro-9 10-bιs(phenylethynyl)anthracene (1 -CI-BPEA) (from Aldπch Chemical Company) and 2 7% rubrene (from (from Aldπch Chemical Company) The particles were latex particles (Seradyn Particle Technology, Indianapolis IN) The dye composition (240-250 mM C-28 thioxene, 8-16 mM 1-CI- BPEA, and 20-30 mM rubrene) was incorporated into the latex beads in a manner similar to that described in U S Patent 5,340,716 issued August 23, 1994 (the 716 patent), at column 48 lines 24-45 which is incorporated herein by reference The dyeing process involved the addition of the latex beads (10% solids) into a mixture of ethylene glycol (65 4%), 2-ethoxyethanol (32 2%) and 0 1 N NaOH (2.3%) The beads were mixed and heated for 40 mm at 95°C with continuous stirring While the beads are being heated, the three chemiluminescent dyes were dissolved in 2- ethoxyethanol by heating them to 95°C for 30 mm with continuous stirring At the end of both incubations the dye solution was poured into the bead suspension and the resulting mixture was incubated for an additional 20 mm with continuous stirring. Following the 20-mιnute incubation, the beads were removed form the oil bath and are allowed to cool to 40^CC ± 10°C The beads were then passed through a 43-mιcron mesh polyester filter and washed The dyed particles were washed using a Microgon (Microgon Inc , Laguna Hills, CA) The beads were first washed with a solvent mixture composed of ethylene glycol and 2-ethoxyethanol (70%/30%). The beads were washed with 500 ml of solvent mixture per gram of beads. This is followed by a 10 % aqueous ethanol (pH 10-11 ) wash The wash volume was 400 mL per gram of beads The beads were then collected and tested for % solid, dye content particle size, signal and background generation Carboxyl acceptor beads prepared above (99mg in 4 5mL water) were added slowly with vortexing to 5 5mL of MAD ammodextran from above, followed by 1 mL of 200mg/mL NHS in 50 mM MES, pH 6, 1 mL of 200 mg/mL EDAC in water, and 450 μL of 1 M HCI, final pH 6 The mixture was incubated overnight at room temperature in the dark, then reacted with 200 mg succmic anhydride in 0.5 mL of DMSO for 30 mm at room temperature Freshly opened Surfact-Amps Tween-20 (Pierce Chemical Company Rockford Illinois) was added and the beads were centrifuged 30 m at 15 000 rpm in a Sorvall RC-5B centrifuge, washed by centrifugation with three 10mL portions of 50 mM MOPS 50 mM EDTA, 0 1 %) Surfact-Amps Tween-20 (Pierce Chemical Company), pH 7 2 and resuspended in 3mL of the same

Monoclonal anti-digoxm Ab (prepared as described above) was purified by ABx resin (Baker Chemical Company Phillipsburg, NJ) and was dialyzed into 0 15 M NaCl, 5mM Na₂HP0₄ pH 7 4 The anti-digoxm Ab was thiolated by mixing 622 μL (4 28mg) with 10 2 uL of SATA (1 25 mg/mL in ethanol, 2 eq ) incubating for 1 hr at room temperature and dialyzing cold against 2x2 L of 150 mM NaCl, 10mM Na₂HPO , 1 mM EDTA pH7 The thioacetylated antibody was deacetylated by adding 62 2 μL of hydroxylamme (1 M H₂NOH, 50 mM MOPS, 25 mM EDTA, pH 7), bubbling with argon and incubating for 1 hr at room temperature The product was applied to a Pharmacia PD-10 column (G-25) and eluted with 50 mM MOPS, 50 mM EDTA, pH 7 2 bubbled with argon After 2 5 mL fore-run, three-1 mL fractions were collected and combined Recovery of antibody was 3 66 mg or 86% by A₂₈o Surfact-Amps Tween-20 (10%) was added to give 0 2% final concentration

A 1 4 mL aliquot of the thiolated antibody above (1 71 mg antibody) was immediately added to 300 μL (10 mg) of maleimidated beads prepared above plus enough 10% Tween-20 to bring final concentration of the mixture to 0 2% The tube was purged with argon and incubated overnight at room temperature in the dark To the above was added 3 4 μL of 1 M HSCH₂COOH in water After 30 mm at room temperature, 6 8 μL of ICH₂COOH (1 M in water) was added After 30 mm 3.5 mL of 0 17M glycme 0 1 M NaCl 0 1 % (v/v) Tween-20, 10 mg/mL BSA, pH 92 was added and the beads were centrifuged (30 mm at 15 000 rpm), incubated for 3hr in 5 mL of the same buffer centrifuged, washed by centrifugation with three-5 mL portions of Buffer C resuspended in 5 mL of Buffer C and stored under refrigeration The size of the beads determined in Buffer C, was 301 +/-56 nm Binding capacity was determined with ¹²⁵l-dιgoxιn and was equivalent to 377 antibody molecules per bead Silicon tetra-t-butyl phthalocyanine was prepared as follows Sodium metal freshly cut (5 Og, 208mmol), was added to 300mL of anhydrous ether in a two-liter 3-necked flask equipped with a magnetic stirrer, reflux condenser, a drying tube and a gas bubbler After the sodium was completely dissolved, 4-t-butyl-1 ,2-dιcyanobenzene (38 64g, 210mmol, from TCI Chemicals, Portland OR) was added using a funnel The mixture became clear and the temperature increased to about 50°C At this point a continuous stream of anhydrous ammonia gas was introduced through the glass bubbler into the reaction mixture for 1 hr The reaction mixture was then heated under reflux for 4 hr. while the stream of ammonia gas continued During the course of the reaction, a solid started to precipitate The resulting suspension was evaporated to dryness (house vacuum) and the residue was suspended in water (400mL) and filtered. The solid was dried (60°C, house vacuum, P₂0₅) The yield of the product (1 ,3- diiminoisoindo ne 42 2g) was almost quantitative This material was used for the next step without further purification To a one-liter, three-necked flask equipped with a condenser and a drying tube was added the above product (18g, 89mmol) and quinoline (200mL Aldπch Chemical Company, St Louis MO) Silicon tetrachloride (11 mL, 95mmol, Aldπch Chemical Company) was added with a syringe to the stirred solution over a period of 10 minutes After the addition was completed, the reaction mixture was heated to 180-185°C in an oil bath for 1 hr. The reaction was allowed to cool to room temperature and concentrated HCI was carefully added to acidify the reaction mixture (pH 5-6) The dark brown reaction mixture was cooled and filtered The solid was washed with 10OmL of water and dried (house vacuum, 60°C, P₂O₅) The solid material was placed in a 1 -liter, round bottom flask and concentrated sulfuric acid (500mL) was added with stirring The mixture was stirred for 4 hr at 60°C and was then carefully diluted with crushed ice (2000g) The resulting mixture was filtered and the solid wad washed with 100mL of water and dried The dark blue solid was transferred to a 1 -liter, round bottom flask, concentrated ammonia (500mL) was added, and the mixture was heated and stirred under reflux for 2 hr , was cooled to room temperature and was filtered The solid was washed with 50mL of water and dried under vacuum (house vacuum, 60°C P₂0₅) to give 12g of product silicon tetra-t-butyl phthalocyanme as a dark blue solid 3-pιcolιne (12g, from Aldπch Chemical Company), tπ-n-butyl amine (anhydrous, 40mL) and tπ-n-hexyl chlorosilane (11.5g) were added to 12g of the above product in a one-liter, three-necked flask, equipped with a magnetic stirrer and a reflux condenser The mixture was heated under reflux for 1 5 hr and then cooled to room temperature The picolme was distilled off under high vacuum (oil pump at about 1mm Hg) to dryness The residue was dissolved in CH₂CI₂ and purified using a silica gel column (hexane) to give 10g of pure product dι-(tπ-n-hexylsιlyl)-sιlιcon tetra-t-butyl phthalocyanme as a dark blue solid (MS [M-H]⁺ 1364 2 absorption spectra methanol 674nm (ε 180,000). toluene 678nm,

¹H NMR (250 MHz, CDCI₃) δ -2 4(m 12H), -1 3(m, 12H), 0.2-0 9 (m, 54H), 1.8(s, 36H), 8 3(d, 4H) and 9 6 (m 8H) was consistent with the above expected product.

Sens-Sav - Sensitizer beads coupled to Streptavidin (2300 Sav/bead). The sensitizer beads were prepared placing 600mL of carboxylate modified beads (Seradyn) in a three-necked, round-bottom flask equipped with a mechanical stirrer, a glass stopper with a thermometer attached to it in one neck, and a funnel in the opposite neck The flask had been immersed in an oil bath maintained at 94+ /-1°C The beads were added to the flask through the funnel in the neck and the bead container was rinsed with 830mL of ethoxyethanol, 1700mL of ethylene glycol and 60mL of 0 1 N NaOH and the rinse was added to the flask through the funnel The funnel was replaced with a 24-40 rubber septum The beads were stirred at 765 rpm at a temperature of 94+ /-1 °C for 40mιn

Silicon tetra-t-butyl phthalocyanme (10 Og) was dissolved in 300mL of benzyl alcohol at 60+/-5°C and 85mL was added to the above round bottom flask through the septum by means of a syringe heated to 120+/-10°C at a rate of 3mL per min. The remaining 85mL of the phthalocyanme solution was then added as described above The syringe and flask originally containing the phthalocyanme was rinsed with 40mL of benzyl alcohol and transferred to round-bottom flask After 15 mm 900mL of deionized water and 75mL of 0 1 N NaOH was added dropwise over 40 m The temperature of the oil bath was allowed to drop slowly to 40+/-10°C and stirring was then discontinued The beads were then filtered through a 43 micron polyester filter and subjected to a Microgon tangential flow filtration apparatus (Microgon Inc Laguna Hills, CA) using ethanol water, 100 0 to 10 90, and then filtered through a 43 micron polyester filter

Sulfo-SMCC (11 55mg) was dissolved in 0 5mL distilled water Slowly, during 10 sec the above solution was added to 5mL of stirring aminodextran (Molecular Probes, Eugene Oregon) solution (12 5 mg/mL in 50mM MOPS, pH 7 2) The mixture was incubated for 1 hr at room temperature

To the stirring solution above was added 5mL of 20mg/mL ( 10Omg) of the sensitizer beads prepared above in distilled water Then, 1 mL of 200mg/mL NHS (prepared fresh in 50mM MES, pH adjusted to 6 0 with 6N NaOH) 200mg EDAC was dissolved in 1 mL distilled water and this solution was added slowly with stirring to the sensitizer beads The pH was adjusted to 6 0 by addition of 450μL of 1 N HCI and the mixture was incubated overnight in the dark A solution of 100mg of succmic anhydride in 0 5mL of DMSO was added to the sensitizer beads and the mixture was incubated for 30 m at room temperature in the dark To this mixture was added 0 13mL 10% Tween-20 bringing the final concentration of Tween-20 to 0 1 % The beads were centrifuged for 45 mm at 15 000 rpm as above The supernatant was discarded and the beads were resuspended in 10mL of buffer (50mM MOPS 50mM EDTA and 0 1 % Tween-20, pH 7 2) The mixture was sonicated to disperse the beads The beads were centrifuged for 30 m as described above the supernatant was discarded and the beads were resuspended This procedure was repeated for a total of three times Then, the beads were resuspended to 40mg/mL in 2 5mL of the above buffer saturated with argon and Tween-20 was added to a concentration of 0 1 % The beads were stored at 4°C Streptavidin was bound to the above beads using 25mg streptavidin for 100mg of beads. 25mg streptavidin (50mg Aaston solid from Aaston, Wellesley, MA) was dissolved in 1 mL of 1 mM EDTA, pH 7.5, and 77μL of 2.5mg/mL SATA in ethanol was added thereto. The mixture was incubated for 30 min at room temperature. A deacetylation solution was prepared containing 1 M hydroxylamine- HCI, 50mM Na₂P0₄₎ 25mM EDTA, pH 7.0. 0.1 mL of this deacetylation solution was added to the above solution and incubated for 1 hr at room temperature. The resulting thiolated streptavidin was purified on a Pharmacia PD10 column and washed with a column buffer containing 50mM MOPS, 50mM EDTA, pH 7.2. The volume of the sample was brought to 2.5mL by adding 1 ,5mL of the above column buffer. The sample was loaded on the column and eluted with 3.5mL of the column buffer. The thiolated streptavidin was diluted to 5mL by adding 1.5mL of 50mM MOPS, 50mM EDTA, 0.1 % Tween-20, pH 7.2. 5mL of the thiolated streptavidin solution was added to 5mL of the sensitizer beads, under argon, and mixed well. The beads were topped with argon for 1 min, the tube was sealed and the reaction mixture was incubated overnight at room temperature in the dark.

To the above beads was added 7.5mL of 50mM MOPS, 50mM EDTA, 0.1 % Tween-20, pH 7.2 to bring the beads to 1 mg/mL. The remaining maleimides were capped by adding mercaptoacetic acid at a final concentration of 2mM. The mixture was incubated in the dark for 30 min at room temperature. The remaining thiols were capped by adding iodoacetic acid at a final concentration of 10mM and the mixture was incubated at room temperature for 30 min in the dark. The beads were centrifuged for 30 min at 15.000 rpm as above for a total of three times.

Example 1

The PCR primer sequences used in the branch migration are set forth in Table 1; the primers have been modified as indicated for the purpose of conducting a "hot start^' procedure as discussed below. Table 1 Specific primers for M tuberculosis rpoB gene mutation detection Forward primers

1779 5'-L-GAGCGGATGACCACCCAGGACNNT-3' (SEQ ID NO 1) where L is Biotin or Digoxigenin

1789 5'- L-CCACCCAGGACGTGGAGGCNNT-3' (SEQ ID NO 2) where L is Biotin or

Digoxigenin Reverse primers

1963a 5'-ACCATGCTCGAGATTACGAG CCGGCACGCTCACGTGACANNA-3 ' (SEQ ID NO 3)

1963b S'-GATCCTAGGCCTCACGTATTCCGGCACGCTCACGTGACANNA-3'

(SEQ ID NO 4) 2044a 5'-ACCATGCTCGAGATTACGAG CAGACCGATGTTGGGCCCCTNNA-3' (SEQ ID NO.5) 2044b δ'-GATCCTAGGCCTCACGTATT CAGACCGATGTTGGGCCCCTNNA-3' (SEQ ID NO.6) 2080a δ'-ACCATGCTCGAGATTACGAG GGGTTGACCCGCGCGTACANNA-3'

(SEQ ID NO 7) 2080b 5'-GATCCTAGGCCTCACGTATT GGGTTGACCCGCGCGTACANNA-3' (SEQ ID NO 8)

Tail 1 5'-ACCATGCTCGAGATTACGAG-3' (SEQ ID NO 9) Tail 2 5'-GATCCTAGGCCTCACGTATT-3' (SEQ ID NO 10) N = etheno dA modification

The position of hybridization of the primers to the rpoB gene sequence is indicated by the primer number The number indicates the target nucleotide complementary to the 5'-end of the primer (shown in bold) In the case of the reverse primers, the position is related to the complementary sequence only, not including tail 1 or tail 2

The positions of the forward and reverse PCR primers are denoted in the detail for the full sequence of the M tuberculosis rpoB gene (GenBank Accession No. U12205) (SEQ ID NO 1 1 ) as depicted in Fig 5 where the single line and double lines under a portion of the gene in bold type is the primer sequence employed and the direction of the arrow indicates forward (->) primers and reverse (<-) primers

The forward PCR primers are 5 -labeled with biotin or digoxigenin (dig) The reverse PCR primers are composed of two parts The 3'-parts of the primers are identical and are complementary to the target DNA (shown in bold) The 5'-parts of the two primers are different and are not related to the target DNA sequence These latter sequences (designated as tail 1 and tail 2) are designed to form the tails of the heteroduplexes, which upon annealing result in the formation of a four stranded DNA structure used in the mutation analysis All forward and reverse primers are also modified at the 3'-end by the addition of two etheno A residues and an additional nucleotide so that a "hot start" procedure may be carried out using an antibody specific for the modifications The 3'-most residue is added for convenience of oligonucleotide synthesis PCR amplification of the rpoB gene sequence PCR amplification of the rpoB gene sequence was carried out using one of two hot start procedures one such procedure was the wax bead-based method using commercially available PCR gems (AmpliWax from Perkm Elmer), the other such procedure involved the use of an anti-etheno A monoclonal antibody, which binds to the primers until the temperature of the reaction medium is raised whereupon the antibody dissociates from the primers and is denatured The choice of primers and conditions for PCR amplification are chosen for specific and efficient production of PCR derived substrates for subsequent branch migration analysis in accordance with the present invention The high GC content of the specific sequence of M tuberculosis rpoB gene also influenced the effectiveness of amplification The amplification conditions described in the following were selected for maximum specificity of the present mutation detection method

PCR amplification of test target was carried out using 5'-bιotιn labeled forward pπmer and two related reverse primers The reference target, wild type, was amplified using the corresponding 5'-dιg labeled forward primer and the same set of reverse pπmers as for the test target amplification PCR amplification with wax bead based hot start was carried out as follows A master mixture (Mix 1 ) containing 10 mM Tris-HCl pH 8 3, 50 mM KCI, 1 5 mM MgCI2, 0 2 mg/ml BSA 200 μM of each of the four dNTPs, and 0 25 μM of each of the primers, was prepared 25 μl of Mix 1 was added to PCR tubes containing a Wax gem, and the tubes were incubated at 80 C, for 2 minutes, to melt the wax gems The reaction tubes were then cooled to room temperature, to form the wax barrier on top of the liquid reaction mixture A second reaction mixture containing 10 mM Tris-HCl pH 8 3 50 mM KCI 1 5 mM MgCI₂, 0 2 mg/ml BSA and 2 5 U/25 μl of Pfu DNA polymerase was also prepared 20 μl of Mix 2 and 5 μl of test or reference target were added to each of the reaction tubes prepared as above

PCR amplification was carried out in Tπo-Thermoblock™ thermocycler (Biometra Inc., Tampa, FI ) The thermocycle program was as follows 4 mm at 95 C followed by 40 cycles of 45 sec at 95 C, and 2 mm at 70 C

PCR amplification using the antibody-based hot start procedure was carried out as follows a master reaction mixture containing 10 mM Tπs-HCI pH 8.3, 50 mM KCI, 1 5 mM MgCI₂, 200 uM of each of four dNTPs, 0 2 mg/ml BSA, 0 5 μM anti etheno A monoclonal antibody (from the Inst fur Zellbiologie, Dr Petra Lorenz), and 1.25 U/25 μl Pfu DNA polymerase was prepared 2 5 μl of test or reference target was added to 22 5 μl of the master reaction mix, in PCR tubes PCR amplification was carried out using conditions similar to the above

Analysis by branch migration and subsequent detection of signals were performed as follows 1 ul of PCR amplification reaction mixture of reference target and 1 μl of test PCR amplification reaction mixture were added to PCR tubes containing 4 μl buffer (100mM Tris-HCl pH8 3, 500mM KCI, 15mM MgCI2 and 2mg/ml BSA) The mixtures were subjected to one cycle of 2 m at 95°C and 30 mm. at 65°C, in a thermocycler 50 μl of a bead mixture (2 5μg of streptavid - coated sensitizer beads and 1 25μg of anti-Dig-coated acceptor beads) were added to each tube, and the tubes were incubated at 37°C for 30 mm and signal was read (3 cycles of 1 sec illumination and 1 sec read) Genotypic detection of πfampin resistance

The results of the analysis of two wild type and two πfampin resistant M tuberculosis clinical isolates are shown in Table 2 The results of analysis using six sets of primer pairs indicate that the signals obtained for wild type and resistant isolates are independent of the size of the amplification product and provide good discrimination between mutant and wild type genotypes The higher signals for mutant 1 and 2 obtained in experiments 4, 5 and 6 reflect higher amplification efficiency as shown on the gel (Native 4-20% gradient polyacrylamide gel from Novex, San Dtego, CA)

Table 2

Detection of mutations in rpoB qene of M tuberculosi s Genomic DNA purified from four clinical isolates was tested usmq 1 two sets of specific primers

Exp# Amplicon size Signal

(base pairs) WT1 WT2 Mut1 Mut2

1 213-bp 39314 34502 386604 454362

2 295-bp 46342 44990 372134 577638

3 331-bp 47320 43540 298460 338174

4 223-bp 40270 40108 987332 937784

5 305-bp 41177 40484 896750 1086440

6 341-bp 43380 27396 1041370 949414

Experiments numbered (Exp#) 1 2 and 3 were carried out using primer sets composed of forward primer 1789 (biotin- or digoxin-labeled) and each of the three reverse primer sets

Experiments numbered 4 5 and 6 were carried out using primer sets composed of forward primer 1779 (biotin- or digoxin-labeled) and each of the three reverse primer sets

Direct genotypic detection of M tuberculosis πfampin resistance using cells grown in culture

M tuberculosis clinical isolates grown in culture were suspended in Buffer B (lOOmMTπs HCI pH 8 3 500mM KCI 15mM MgCI2 and 2mg/ml BSA) and heat inactivated by boiling for 30 m at 95°C Direct analysis was achieved following sonication, (12-15 pulse on Branson Sonifier 450 ), boiling ( 98°C for 15-mιn , using a thermocycler) or treatment in a microwave (45 sec to 1-mιn at high power setting). The direct analysis of 10 M. tuberculosis clinical isolates, obtained using cell suspensions pretreated as detailed above demonstrated direct genotypic detection (Table 3).

Comparison of results obtained by culture-based phenotypic determination of πfampin susceptibility (reference laboratory) and genotypic detection using the branch migration method described herein revealed a single discrepancy: A clinical isolate determined as antibiotic susceptible by the culture-based test, scored as resistant by the genotypic method. It is possible that the positive result obtained by the branch migration method reflects the presence of a silent mutation, as was previously shown (Mutations in the rpoB gene of M. tuberculosis that interfere with PCR-Single strand conformation polymorphism analysis for rifampin susceptibility testing (Kim B-J, et al J. Clinical Microbiology 35, 492, 1997). The high specificity of the branch migration genotypic detection method assures the detection of all resistant isolates, a feature most important for the control of Mycobacterial infection.

Table 3

Results obtained with heat killed M. tuberculosis isolates: heat killed cells were disrupted by sonication

Phenotype

Isolate RLU-1^* RLU-2^** genotype (Ref.

Lab)

1 16824 8840 wt susceptible

2 755440 1434180 mutant resistant

3 17040 9724 wt susceptible

4 626394 1495320 mutant resistant

5 11346 10980 wt susceptible

6 1065130 1363690 mutant resistant

7 1112210 1227160 mutant resistant

8 1237660 1421840 mutant susceptible

9 1024550 1047470 mutant resistant

10 29212 10208 wt susceptible

RLU-1^* = relative luminescence unit for M. tuberculosis cells heat killed Buffer A RLU-2" = relative luminescence unit for M. tuberculosis cells heat killed in Buffer B Example 2

Detection of genotype of M tuberculosis associated with πfampin resistance utilizing normalization method

As shown in previous examples the branch migration method was effective in the detection of rpoB gene alteration Signals were indicative of sequence alteration of test sequence relative to reference sequence Low signals indicated identity of the test sequence to reference sequence However, since the signal was related also to the amount of test target relative to the reference target, low input test target may also result in low signal, which may lead to false determination of mutant genotype as a wild type genotype

In accordance with the present invention, the following experiments demonstrate a method for normalization of the signals relative to input test target As explained above, the normalization method is based on formation of stable four stranded DNA structures when test target amplification products are mixed with similarly produced products of amplification of non-relevant reference sequence In so far as the two sequences are not related signals are produced from all test samples, regardless of the specific genotype The ratio of signals produced with relevant reference sequence to those produced with non-relevant reference sequence are the normalized signals and represent test genotype regardless of input target sequence

As shown below 46 samples of M tuberculosis genomic DNA purified from clinical isolates were tested in a ' blinded" fashion for rpoB genotype The signals were produced as described in the detailed protocols The jest DNA amplification products were tested against relevant wild type target genomic DNA and non- relevant reference sequence, in this case amplification products of the human cystic fibrosis gene Amplification of the non-relevant reference sequence was carried out with reverse primers designed for branch migration analysis and composed of a 3 target specific portion and 5' tails, which were the same as those used for the M tuberculosis rpoB analysis This feature is important for the ability of forming four stranded structures between the test and non-relevant amplification products

The results of the blinded study are summarized in Table 4 The ratio of signal obtained with relevant reference to signal obtained with non-relevant reference clearly differentiate mutant and wild type isolates The mutant and wild type genotype determinations by this method are in full agreement with phenotypic determination of the clinical isolates as πfampin resistant or sensitive

PCR amplification of non-relevant target was carried out using the antibody- based hot start procedure as described above All of the reagents were the same as stated above except the target was cystic fibrosis exonl 1 (wild type) and the primers were 5'-dιg labeled forward primer and a mixture of reverse primers modified with the same taιl-1 and taιl-2 as for M tuberculosis as described above The primers are identified in the CFTR Gene, Exon 11 Sequence below Analysis and detection were performed as follows (1 ) For genotype determination

1 μl of PCR amplification reaction mixture of reference target and 1 μl of test PCR amplification reaction mixture were added to PCR tubes containing 4 μl buffer (100mM Tπs-HCI pH8 3 500mM KCI 15mM MgCI2 and 2mg/ml BSA) The mixtures were subjected to one cycle of 2 m at 95°C and 30 m at 65°C, in a thermocycler 50 μl of a bead mixture (2 5μg of streptavidm-coated sensitizer beads and 1 25 μg of anti-Dig-coated acceptor beads) were added to each tube, and the tubes were incuoated at 37°C for 30 m and signal was read (3 cycles of 1 sec illumination and 1 sec read) (2) For DNA Confirmation 1 μl of PCR amplification reaction mixture of PCR amplified non-relevant target of wild type CF at 1/10 dilution and 1 μl of test PCR amplification reaction mixture were added to PCR tubes containing 4 μl buffer (100mM Tπs-HCI pH8 3, 500mM KCI, 15mM MgCI2 and 2mg/ml BSA) The mixtures were subjected to one cycle of 2 m at 95°C and 30 m at 65°C in a thermocycler 50 μl of a bead mixture (2.5 μg of streptavidm-coated sensitizer beads and 1 25 μg of anti-Dig- coated acceptor beads) were added to each tube, and the tubes were incubated at 37°C for 30 mm and signal was read (3 cycles of 1 sec illumination and 1 sec read)

Blind studies of M tuberculosis rpoB gene mutation detection 46 samples of M. tuberculosis genomic DNA were tested for the genotype indicative of πfampin resistance using an assay in accordance with the present invention followed by detection with anti-dig Ab coated acceptor chemiluminescent beads and streptavidin coated sensitizer beads The results are summarized in Table 4.

Table 4

CFTR Gene. Exon 1 1 Sequence

1 atatacccat aaatatacac atattttaat ttttggtatt ttataattat tatatgggta tttaratgtg tataaaatta aaaaccataa aatattaata

51 tatttaatga tcattcatga cattttaaaa attacaggaa aaatttacat ataaattact agtaagtact gtaaaatttt taatgtcctt tttaaatgta

101 ctaaaatttc agcaatgttg tttttgacca actaaataaa ttgcatttga gattttaaag tcgttacaac aaaaactggt tgatttattt aacgtaaact

151 aataatggag atgcaatgtt caaaatttca actgtggtta aagcaatagt ttattacctc tacgttacaa gttttaaagt tgacaccaat ttcgttatca f2-B Biotin-5 ' -tag aaggaagatg tgcctttca-3 ' f2-D Digoxin-5 ' -tag aaggaagatg tgcctttca-3"

201 gtgatatatg attacattaq aaαqaaqatq tqcctttcaa attcagattg cactatatac taatgtaatc ttccttctac acggaaagtt taagtctaac

251 agcatactaa aagtgactct ctaattttct atttttggta ataggacatc tcgtatgatt ttcactgaga gattaaaaga taaaaaccat tatcctgtag 542

301 tccaagtttg cagagaaaga caatatagtt cttGGAqaaq gtggaatcac aggttcaaac gtctctttct gttatatcaa gaacctcttc caccttagtg

551 553 560 351 actgagtgga GGTcaaCGAq caagaatttc tttagcaAGG tgaataacta tgactcacct ccagttgctc grtcttaaag aaatcgttcc acttattgat

401 attattggtc tagcaagcat ttgctgtaaa tgtcattcat gtaaaaaaat taataaccag atcqttc ta aacαacattt acaqtaaqta cattttttta rltl 3'-cgttcgta aacgacattt acag

-ttatgcactccggatcctag-5 ' rlt2 3 ' -cgttcgta aacgacattt acag

-gagcattagagctcgtacca-5 ' 451 tacagacatt tctctattgc tttatattct gtttctggaa ttgaaaaaat atgtctgtaa agagataacg aaatataaga caaagacctt aactttttta

501 cctggggttt tatggctagt gggttaagaa tcacatttaa gaactataaa ggaccccaaa ataccgatca cccaattctt agtgtaaatt cttgatattt

551 taatggtata gtatccagat ttggtagaga ttatggttac tcagaatctg attaccatat cataggtcta aaccatctct aataccaatg agtcttagac 601 tgcccgtatc ttgg 3' 614 (SEQ ID NO: 12) acgggcatag aacc 5' (SEQ ID NO: 13)

The f2/rl primer ser flanks 173 bases of the CFTR Exon 11 sequence, resulting in an amplicon which includes 217 bases from Exon 11 and 20 bases from the reverse primer tails, for a total of 237 bp. The above discussion includes certain theories as to mechanisms involved in the present invention These theories should not be construed to limit the present invention in any way, since it has been demonstrated that the present invention achieves the results described

The above description and examples fully disclose the invention including preferred embodiments thereof Modifications of the methods described that are obvious to those of ordinary skill in the art such as molecular biology and related sciences are intended to be within the scope of the following claims

Claims

WHAT IS CLAIMED IS

1 A method for detecting the presence of a difference between two related nucleic acid sequences, said method comprising (a) forming a complex comprising both of said nucleic acid sequences in double stranded form, wherein said complex comprises at least one pair of non- complementary strands and each of said non-complementary strands within said complex has a label,

(b) subjecting said complex to conditions wherein, if a difference between said two related nucleic acid sequences is present, strand exchange in said complex ceases and wherein if no difference between said two related nucleic acid sequences is present strand exchange in said complex continues until complete strand exchange occurs,

(c) detecting a first signal from the association of said labels as part of said complex, the association thereof being related to the presence of said difference,

(d) forming a complex comprising said nucleic acid sequence suspected of comprising a difference in double stranded form and a predetermined amount of a non-relevant reference polynucleotide in double stranded form, wherein said complex comprises at least one pair of non-complementary strands and each of said non-complementary strands within said complex has a label,

(e) detecting a second signal from the association of said labels as part of said complex of step (d),

(f) determining a ratio of said first signal to said second signal, and (g) relating said ratio to the presence of said difference

2 A method according to Claim 1 wherein said difference is a mutation

3 A method according to 1 wherein said nucleic acid sequences are DNA.

4 A method for detecting a mutation within a target nucleic acid sequence, said method comprising

(a) forming from said target sequence a tailed target partial duplex A' comprised of a duplex of two nucleic acid strands of said target sequence, a label and at one end of said duplex, two non-complementary oligonucleotides, one linked to each of said strands,

(b) providing in combination said tailed target partial duplex A and a tailed first reference partial duplex B' lacking said mutation having a label as a part thereof, wherein said tailed reference partial duplex B' is comprised of two nucleic acid strands each of said strands being complementary respectively, to a strand in said tailed target partial duplex A' but for the possible presence of a mutation and wherein said labels are present in non-complementary strands of said tailed target and tailed reference partial duplexes, respectively, (c) subjecting said combination to conditions wherein, if a mutation is present, strand exchange in said complex ceases and wherein, if no mutation is present, strand exchange in said complex continues until complete strand exchange occurs,

(d) detecting a first signal by means of said labels resulting from the formation of a complex between said tailed partial duplexes, the formation thereof being directly related to the presence of said mutation,

(e) providing in combination said tailed target partial duplex A and a tailed second reference partial duplex B' having a label as a part thereof, wherein said tailed second reference partial duplex B' is comprised of two nucleic acid strands of a non-relevant polynucleotide wherein said labels are present in non-complementary strands of said tailed target and tailed second reference partial duplexes respectively,

(f) subjecting said combination to conditions wherein a complex comprising a four-stranded structure is formed, (g) detecting a second signal by means of said labels resulting from the formation of said complex of step (f)

(h) determining a ratio of said first signal to said second signal, and

(i) relating said ratio to the presence of said mutation

5 A method according to Claim 4 wherein said nucleic acid sequences are DNA

6 The method of Claim 4 wherein said tailed reference partial duplex B' is provided in said combination by forming said tailed reference partial duplex B' in the same reaction medium as that used for step (a)

7 The method of Claim 6 wherein forming said tailed target partial duplex A' and said tailed reference partial duplex B' is carried out simultaneously

8 The method of Claim 4 wherein said labels are independently selected from the group consisting of oligonucleotides, enzymes, dyes, fluorescent molecules, chemilummescers, coenzymes, enzyme substrates, radioactive groups, small organic molecules and solid surfaces

9 The method of Claim 4 wherein said non-complementary oligonucleotides each have from 15 to 60 nucleotides

10 A method of detecting a mutation within a target nucleic acid sequence, said method comprising

(a) amplification of said target sequence by polymerase chain reaction, using primers P1 and P2 to produce an amplicon AA, wherein one of said primers P1 and P2 comprises a label and wherein said primer P1 is comprised of a 3'-end portion Pa that can hybridize with said target sequence and 5'-end portion B1 that cannot hybridize with said target sequence, (b) extending a primer P3 by chain extension along one strand of amplicon AA to produce a tailed target partial duplex A', wherein said primer P3 is comprised of said 3'-end portion Pa and a 5'-end portion A1 that cannot hybridize to said target sequence or its complement (c) amplification of a first reference nucleic acid sequence, using said primer P2 and said primer P3, by polymerase chain reaction to produce amplicon BB, said first reference sequence being identical to said target sequence but lacking a possible mutation, wherein said primer P2 comprises a label when said primer P2 in step (a) above comprises a label and said primer P3 comprises a label when said primer P1 in step (a) above comprises a label,

(d) extending said primer P1 by chain extension along one strand of amplicon BB to produce a tailed reference partial duplex B',

(e) allowing said tailed target partial duplex A' to bind to said tailed first reference partial duplex B', and (f) detecting a first signal said labels as a result of the formation of a complex between said tailed partial duplexes, the formation thereof being directly related to the presence of said mutation,

(g) amplification of a second reference nucleic acid sequence, using a set of primers, by polymerase chain reaction to produce amplicon CC, said reference sequence being non-relevant to said target sequence, wherein said primers comprise priming sequences for said second reference and respectively, said 5'-end portion B1 and said 5 -end portion A1 wherein one of said primers comprises a label,

(h) extending said primer P1 by chain extension along one strand of amplicon CC to produce a tailed reference partial duplex C,

(i) allowing said tailed target partial duplex A' to bind to said tailed reference partial duplex C, and

(j) detecting a second signal from said labels as a result of the formation of a complex comprising a four-stranded structure between said tailed partial duplexes of step (i), (k) determining a ratio of said first signal to said second signal, and (I) relating said ratio to the presence of said mutation.

11. A method according to Claim 10 wherein said nucleic acid sequences are DNA.

12. A method according to Claim 10 wherein said labels are independently selected from the group consisting of oligonucleotides, enzymes, dyes, fluorescent molecules, chemiluminescers, coenzymes, enzyme substrates, radioactive groups, small organic molecules and solid surfaces.

13. The method of Claim 10 wherein said amplification of step (c) is carried out in the same reaction medium as that used for step (a).

14. The method of Claim 1 1 wherein said amplification of step (c) is carried out simultaneously with the amplification of step (a).

15. The method of Claim 10 wherein the label of primer P2 in step (c) is different than the label of primer P2 in step (a).

16. The method of Claim 13 wherein said labels are independently selected from the group consisting of oligonucleotides, enzymes, dyes, fluorescent molecules, chemiluminescers, coenzymes, enzyme substrates, radioactive groups, small organic molecules and solid surfaces.

17. The method of Claim 10 wherein said nucleic acid is DNA.