WO2001038580A2

WO2001038580A2 - Nucleic acid probe arrays

Info

Publication number: WO2001038580A2
Application number: PCT/US2000/032131
Authority: WO
Inventors: Jonathan M. Rothberg; Joel S. Bader
Original assignee: Curagen Corporation
Priority date: 1999-11-26
Filing date: 2000-11-27
Publication date: 2001-05-31
Also published as: WO2001038580A9; AU783841B2; EP1234058A2; CA2392474A1; JP2003515149A; WO2001038580A3; AU1792701A

Abstract

Disclosed are nucleic acid probe arrays and methods of identifying and sequencing nucleic acids in a population of nucleic acids using the arrays. The method is preferably performed by annealing a nucleic acid template to an anchor primer attached to a durface of the array. At least one anchor in the array has a sequence complementary to sequences at the 5' and 3' termini of a target nucleic acid.The annealed linear target nucleic acid is circularized using one or two ligation reactions. In one embodiment, one liggation is issued. Annealing of of the linear nucleic acid results in juxtaposition of the 5' and 3' termini of the target nucleic acid on the anchor primer. Addition of a ligase results in circulization of the target nucleic acid. This circularized nucleic acid is a template for extension of the anchor primer in a rolling circle amplification reaction. An extended anchor primer containing multiple copies of a sequence complementary to the circular mucleic acid is also referred to herein as a anchor primer nucleid acid-nucleic acid concatamer. The presence of multiple copies of the complementary sequence facilitates detection of the nucleic acid. Thus, the method provides for a highly sensitive method of detecting a desired nucleic acid attached at a discrete location on the array.

Description

NUCLEIC ACID PROBE ARRAYS

Field of the Invention The invention relates generally to nucleic acids and more particularly to arrays of nucleic acids for identifying nucleic acids in a population of nucleic acids.

Background of the Invention Many diseases are associated with particular DNA sequences. The DNA sequences are often referred to as DNA sequence polymorphisms to indicate that the DNA sequence associated with a diseased state differs from the corresponding DNA sequence in non-afflicted individuals. DNA sequence polymorphisms can include, e.g., insertions, deletions, or substitutions of nucleotides in one sequence relative to a second sequence. An example of a particular DNA sequence polymorphism is 5'-ATCG-3', relative to the sequence 5'-ATGG-3'. The first nucleotide 'G' in the latter sequence has been replaced by the nucleotide 'C in the former sequence. The former sequence is associated with a particular disease state, whereas the latter sequence is found in individuals not suffering from the disease. Thus, the presence of the nucleotide sequence '5-ATCG-3' indicates the individual has the particular disease. This particular type of sequence polymorphism is known as a single-nucleotide polymorphism, or SNP, because the sequence difference is due to a change in one nucleotide. Techniques that allow for the rapid detection of as little as a single DNA base change are therefore important methods for use in genetic analysis. Because the size of the human genome is large, on the order of 3 billion base pairs, techniques for identifying polymorphisms must be sensitive enough to specifically identify the sequence containing the polymorphism in a potentially large population of nucleic acids. Typically a DNA sequence polymorphism analysis is performed by isolating DNA from an individual, manipulating the isolated DNA, e.g., by digesting the DNA with restriction enzymes and/or amplifying a subset of sequences in the isolated DNA. The manipulated DNA is then examined further to determine if a particular sequence is present.

Techniques for analyzing DNA sequences include gel-based electrophoretic analysis, scanning tunnel electron microscopy, sequencing by hybridization, and solid substrate-based nucleic acid analyses. These solid supports can include, e.g., glass surfaces, plastic microtiter plates, and plastic sheets. The substrates typically contain a plurality of linked oligonucleotides or polynucleotides that can be, e.g., adsorbed or covalently attached to the support. Substrate-based nucleic acid analyses can include applying a sample nucleic acid known or suspected of containing a particular sequence polymorphism to an array of probes attached to the solid substrate. The nucleic acids in the population are allowed to hybridize to complementary sequences attached to the substrate, if present. Hybridizing nucleic acid sequences are then detected in a detection step.

Solid support matrix-based hybridization and sequencing methods can require a high sample-DNA concentration and can be hampered by the relatively slow hybridization kinetics of nucleic acid samples with immobilized oligonucleotide probes. Often, only a small amount of template DNA is available.

Summary of the Invention The invention is based in part on the discovery of a sensitive method for generating a clonally amplified nucleic acid at a discrete location in an array of nucleic acids. The method is preferably performed by annealing a nucleic acid template to an anchor primer attached to a surface of the array. At least one anchor primer in the array has a sequence complementary to sequences at the 5' and 3' termini of a target nucleic acid. Annealing of a desired target nucleic acid to the anchor primer results in juxtaposition, or near juxtaposition, of the 5' and 3' ends of the linear target nucleic acid.

The annealed linear target nucleic acid is circularized using one or two ligation reactions. In one embodiment, one ligation is used. Annealing of the linear nucleic acid results in juxtaposition of the 5' and 3' termini of the target nucleic acid on the anchor primer. Addition of a ligase results in circularization of the target nucleic acid. This circularized nucleic acid is a template for extension of the anchor primer in a rolling circle amplification reaction. An extended anchor primer containing multiple copies of a sequence complementary to the circular nucleic acid is formed. A nucleic acid containing an anchor primer covalently linked to two or more copies of a sequence complementary to the target nucleic acid is also referred to herein as a anchor primer nucleic acid -nucleic acid concatamer.

The presence of multiple copies of the complementary sequence facilitates detection of the nucleic acid. Thus, the method provides for a highly sensitive method of detecting a desired nucleic acid attached at a discrete location on the array. Because only a few anchor primers in the array will typically be extended in a given reaction, arrays containing high densities of anchor primers can be prepared. Thus, the methods and compositions of the invention provide high density arrays in which desired nucleic acids can be detected with high sensitivity.

In an alternative embodiment, two ligation events occur. The 5' and 3' termini of the annealed target nucleic acids are separated by a gap. An oligonucleotide complementary to the gap is added. Ligation of the termini of the annealed target nucleic acid to the gap oligonucleotide results in formation of a covalently closed circular molecule that can act as a template for rolling circle amplification.

When either one or two ligation events are used, extension of the anchor primer using as a template the circular nucleic acid molecule results in clonally amplified product at a discrete location in the substrate.

The requirement of one or two ligation reactions increases allows for increased sensitivity in identifying a desired nucleic acid molecule because it requires a perfect pairing between the terminal nucleotides of the target nucleic acid molecule and the corresponding nucleotides in the anchor primer. Ligase molecules preferentially ligate substrates in which the 3' and 5' terminal nucleotides are base-paired to complementary nucleotides. Accordingly, duplexes of a target nucleic acid and an anchor primer containing a mismatched oligonucleotide at or near the termini of the target nucleic acid are not substrates for the ligase and therefore do not produce a covalently closed circle.

Thus, the invention provides a highly sensitive method for using an array to identify and clonally amplify a target nucleic acid in a population of nucleic acid molecules.

A preferred polynucleotide anchor primer includes a first region, a second region, and a third region. The first region includes a sequence complementary to a 5' region of a target nucleic acid sequence, and the second region includes a sequence complementary to a 3' region of the target nucleic acid sequence. The first and second regions can each be 4-25, 4- 20, 8-20, 10-18, or 12-16 nucleotides in length. In particular embodiments, the first region or second region, or both, is, e.g., 4, 6, 8, 10, 12, 16, 18, 20, or 25 nucleotides in length. The third region can be, e.g., 4-20, 6-18, 8-16, or 10-14 nucleotides in length. In particular embodiments, the third region is, e.g., 4, 6, 8, 10, 12, 14, 16, 18, or 20 nucleotides in length. The target nucleic acid sequence can include, e.g., a restriction fragment produced by digesting a starting population of nucleic acids with one or more restriction enzymes.

In some embodiments, the first region includes a sequence complementary to some or all of the recognition sequence of a first restriction endonuclease, and a region complementary to a sequence of a target nucleic acid sequence. The complementary region is preferably 3' to the recognition sequence for the first restriction endonuclease in the target nucleic acid sequence.

The first region may in addition include a query nucleotide complementary to a nucleotide defining a sequence polymorphism in a target nucleic acid. Preferably, the query nucleotide is at the 3' terminus of the first region, i.e., at the 3' terminus of the polynucleotide.

In some embodiments, the third region includes a sequence complementary to some or all of a recognition sequence of a second restriction endonuclease, and a region complementary to a sequence of the target nucleic acid sequence. The complementary region is preferably located 5' to the recognition sequence for the second restriction endonuclease in the target nucleic acid sequence.

The first and second restriction enzymes can be identical or different. In addition, the restriction enzymes can be, e.g., type II or type IIS restriction enzymes.

In some embodiments, the oligonucleotide can be, e.g., 4-25, 8-20 or 6-12 nucleotides in length. For example, in particular embodiments, the oligonucleotide can be, e.g., 4, 6, 8, 10, 12, 16, 18, 20, or 25 nucleotides in length.

The target nucleic acid may contain, or be suspected of containing, a sequence polymorphism. The polymorphism can in some embodiments be located within, e.g., 200, 100, 75, 50, 25, or 15 nucleotides of the 5' or 3' terminus of the target nucleic acid sequence, or at the 5' or 3' terminus of the target nucleic acid. In a further aspect, the invention provides an array of oligonucleotide anchor primers

(either of identical or differing sequences). The array includes a support having a first surface linked thereto a plurality of oligonucleotide anchor primers attached to the first surface of the solid support. The anchor primers attached to the surface include a first region complementary to a 5' region of a target nucleic acid sequence, a second region complementary to a 3' region of the target nucleic acid sequence, and an optional third region located between the first region and the second region. Each of the different oligonucleotides is preferably attached to the surface of the solid support in a different predetermined region and has a different predetermined sequence.

Attachment of the anchor primer to the surface can occur before, during, or subsequent to extension of the annealed anchor primer. Thus, in one embodiment, one or more anchor primers are linked to the solid substrate, after which the anchor primer is annealed to a target nucleic acid and extended in the presence of a polymerase. Alternatively, in a second embodiment, an anchor primers is first annealed to a target nucleic acid, and a 3 'OH terminus of the annealed anchor primer is extended with a polymerase. The extended anchor primer is then linked to the solid substrate. By varying the sequence of anchor primers, it is possible to specifically amplify distinct target nucleic acids present in a population of nucleic acids.

In some embodiments, the anchor primer is 12-100, 18-50 or 24-40 nucleotides in length. In particular embodiments the anchor primer is, e.g., 12, 18, 24, 40, or 50 nucleotides in length. The aπay can include at least 100, or even 1,000, 10,000, 100,000, or 1,000,000, or 10,000,000 or more different oligonucleotides attached to a solid support.

Preferably, each of the predefined regions is physically separated from each of the other predetermined regions, e.g., the defined regions can be separated by 1-400 μm, 40-150 μm, or 100-150 μm.

In another aspect, the invention provides a method of determining the sequence of a nucleic acid. The method includes providing a substrate having a plurality of different anchor primers, e.g., the anchor primers described above. In some embodiments, the plurality includes anchor primers of known sequence at known locations on the array. The anchor primers include a first region complementary to a 5' region of a target nucleic acid sequence, a second region complementary to a 3' region of the target nucleic acid sequence, and, optionally, a third region lying between the first and second region.

One or more of the anchor primers on the array are contacted with a population of circular target nucleic acid molecules. The target circular nucleic acids can be open circles or closed circles.

In some embodiments, the anchor primers on the substrate are contacted with a circular nucleic acid having 5' and 3' regions complementary to the first and third regions of an anchor primer having the first and third regions.

In some embodiments, the circular target nucleic acid is formed by annealing an anchor primer in the array with a linear target nucleic acid having 5' and 3' termini under conditions which allow the 5' and 3' termini of the target nucleic acid to anneal to complementary sequences in an anchor primer in the array. The annealed termini of the target nucleic acid are then contacted with a gap oligonucleotide complementary to the third region, (which can also be called a gap region) of the anchor primer and a ligase under conditions sufficient allow for ligation of the 5' and 3' termini of the target nucleic acid to the gap oligonucleotide. This results in formation of a closed circular target nucleic acid.

In some embodiments, sequence homologous to the closed circular molecule are amplified prior to determining their nucleotide sequence. Amplification take place, e.g., by extending the 3' OH terminus of an anchor primer annealed to a closed circular molecule. Extension occurs with a polymerase under conditions that allow for formation of a polynucleotide product complementary to one or more copies of the covalently closed circular target nucleic acid. The polynucleotide product is identified, thereby determining the sequence of the circularized target nucleic acid.

The target nucleic acid can contain a sequence polymorphism, e.g., a single nucleotide sequence polymorphism. Preferably, the single nucleotide polymorphism is complementary to a query nucleotide in said first region of said anchor primer, e.g., a query nucleotide at the 3' terminus of the anchor primer. In some embodiments, the covalently closed circular nucleic acid is formed by contacting one or more of the anchor primers on the array with a linear target nucleic acid under conditions that allow the 5' and 3' termini of the target nucleic acid to anneal to the anchor primers. The 5' and 3' termini may in some embodiments by separated by a third, "gap region" of at least 4 nucleotides in the anchor primer. If the gap region is present, the 3' terminus of the target nucleic acid is then extended with a polymerase under condition sufficient to form an extended product whose 3' terminal nucleotide abuts the 5' terminus of the annealed target nucleic acid.

Next, the 3' terminal nucleotide of the extended product is ligated to the 5' terminus of the annealed target nucleic acid, thereby forming a closed circular molecule. The target molecule can come from any population of nucleic acid molecules, e.g., a population of DNA or RNA molecules.

In some embodiments, the anchor primers on the substrate are contacted with a linear target nucleic acid under conditions to allow the 5' and 3' termini of the target nucleic acid to anneal to the anchor primers. The 5' and 3' termini, when annealed to the anchor primers, are separated by a gap region of at least 6 nucleotides on the anchor primer.

Next, the annealed termini of the target nucleic acid are contacted with a gap oligonucleotide and a ligase. The gap oligonucleotide is complementary to the gap region of the anchor primer. The ligase is added under conditions sufficient to allow for ligation of the 5' and 3' termini of the target nucleic acid to the gap oligonucleotide, thereby forming a covalently closed circular molecule that includes the nucleotide sequence of the target nucleic acid and the gap oligonucleotide. The nucleotide sequence of at least a portion of the covalently closed circular molecule is determined, thereby determining the sequence of the target nucleic acid. In some embodiments, the circular (closed or open) target nucleic acid is sequenced directly, e.g., by extending the anchor primer using the annealed target nucleic acid sequence as a template. The target nucleic acid to be sequenced may contain, or be suspected of containing, a sequence polymorphism. The polymorphism can in some embodiments be located within, e.g., 200, 100, 75, 50, 25, or 15 nucleotides of the 5' or 3' terminus (prior to of the target nucleic acid sequence, or at the 5' or 3' terminus of the target nucleic acid. In some embodiments, the polymorphism is at a nucleotide complementary to a query nucleotide present in the 3' terminal region of the anchor primer, e.g., at the 3' terminus of the anchor primer. In other embodiment, the circularized target nucleic acid is amplified prior to determining its nucleotide sequence. Amplification can occur, e.g., by extending an annealed anchor primer annealed to a circular target molecule to generate a concatemer of sequences complementary to the circular target molecule. At least a portion of the amplified polynucleotide product is then identified, thereby determining the sequence of the circularized target nucleic acid.

The linear target nucleic acid molecule can contain, e.g., a sequence polymorphism such as a single nucleotide polymorphism.

In a further aspect, the invention provides a method of determining the sequence of a nucleic acid. The method includes providing a substrate having a plurality of different anchor primers of known sequence at known locations. The anchor primers include a first region complementary to a 5' region of a target nucleic acid sequence, a second region complementary to a 3' region of the target nucleic acid sequence, and, optionally, a third region located between the first region and second region. The substrate is then contacted with a linear target nucleic acid under conditions to allow the 5' and 3' termini of the target nucleic acid to anneal to the anchor primers. In preferred embodiments, the 5' and 3' termini are separated by a region corresponding to the second region (which is also known as a gap region) of at least 6 nucleotides on the anchor primer. Next, the 3' terminus of the target nucleic acid is extended with a polymerase under condition sufficient to form an extended product whose 3' terminal nucleotide abuts the 5' terminus of the annealed target nucleic acid. The 3' terminal nucleotide of the extended product is ligated to the 5' terminus of the annealed target nucleic acid, thereby forming a covalently closed circular molecule comprising the nucleotide sequence of the target nucleic acid and the gap oligonucleotide. The nucleotide sequence of at least a portion of the covalently closed circular molecule is determined as described above.

Also provided is a method for comparing the relative amount of a target nucleic acid in a first and second population of nucleic acid molecules. The method includes providing one or more or more nucleic acid anchor primers linked to a solid support, as well as a first plurality of circular single-stranded nucleic acid templates derived from the first population of nucleic acid molecules and a second plurality of open or closed circular single-stranded nucleic acid molecules derived from the second population of nucleic acid molecules. An effective amount of a nucleic acid anchor primer is then annealed to at least one of the single-stranded circular templates derived from the first plurality of circular single-stranded nucleic acid molecules, to yield a first primed single-stranded circular template. An effective amount of the nucleic acid anchor primers is also annealed to at least one of the single-stranded circular templates from the second plurality of circular single-stranded nucleic acid molecule to yield a second primed single-stranded circular template. The first and second primed anchor primer-circular template complexes are used to determine the sequence of the first and second nucleic acid molecules. In some embodiments, the sequences are determined directly. In other embodiments, the sequences are determined after amplifying the target nucleic acids, e.g., after amplification with a polymerase to generate multiple copies of the circular nucleic acid template derived from the first and second populations of nucleic acid molecules. The sequence of the target nucleic acids can be determined by annealing an effective amount of a sequencing primer to the first and second circular nucleic acid templates to yield first and second primed sequencing primer-circular nucleic acid template complexes. The complexes are then extended to yield a first and second sequencing product associated with the first and second primed sequencing primer-circular nucleic acid template complexes. Levels of the first and second sequencing products are then compared. The relative level of the first and second sequencing products indicates the relative level of the target nucleic acid in the first and second population of nucleic acid sequences.

The first and second population of nucleic acid molecules can be, e.g., genomic DNA sequences, or derived from RNA sequences. The population of nucleic acid sequences can include, e.g., a first population of nucleic acids is from a cell population that has been exposed to a stimulus and the second population of nucleic acids is from the same cell population that has not been exposed to the stimulus. Stimuli can include, e.g., a physical stimulus (e.g., heat), a mitogen, and a ligand for a receptor expressed by cells in the cell population. In addition, the first and second population of nucleic acids can be obtained from cells of two different individuals.

If desired, levels of nucleic acid sequences from multiple populations can be compared, e.g., nucleic acid molecules in 3, 5, 10, 25, 50, 100, 1,000. or 10,000 or more populations. In a still further aspect, the invention provides a method for detecting an RNA molecule in a population of RNA molecules. The method includes providing a population of cDNA molecules and a substrate having a plurality of different anchor primers of known sequence at known locations. The anchor primers include a first region complementary to a 5' region of a target nucleic acid sequence, a second complementary to a 3' region of the target nucleic acid sequence, and, optionally, a third region (also known as a gap region) located between the first and third regions. The substrate is then contacted with a linear target nucleic acid in the population of cDNA molecules under conditions sufficient to allow the 5' and 3' termini of the target nucleic acid to anneal to the anchor primers. The 5' and 3' termini are preferably separated by a gap region of at least 6 nucleotides on the anchor primer. The annealed termini are then contacted with a gap oligonucleotide complementary to the gap region of the anchor primer and a ligase under conditions sufficient allow for ligation of the 5' and 3' termini of the target nucleic acid to the gap oligonucleotide. As a result, a covalently closed circular molecule including the nucleotide sequence of the target nucleic acid and the gap oligonucleotide is formed. The nucleotide sequence of at least a portion of the covalently closed circular molecule is then determined, thereby detecting the RNA molecule. The details of one or more embodiments of the invention are set forth in the accompanying description below. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications cited in this specification are incorporated by reference.

Brief Description of the Drawings Figs. 1 A and IB are schematic drawings of an anchor primer according to the invention. FIG. 2 is a schematic drawing of a second anchor primer according to the invention.

FIG. 3 is a schematic drawing of a third anchor primer according to the invention.

FIG. 4 is a schematic drawing of rolling circle based amplification product linked to an anchor primer. Detailed Description of the Invention

The invention provides arrays of polynucleotide and oligonucleotide anchor probes that can be used, e.g., in identifying and sequencing nucleic acids. Structure of Anchor Probes

An anchored primer according to the invention is shown in FIG. 1. An anchor primer 100 is attached at its 5' end to a substrate 102. The anchor primer includes regions 104, 106, and 108, which are contiguous with one another.

Also shown in FIG. 1 is a linear target nucleic acid 1 10 having region 1 14 at its 5' terminus and region 112 at its 3' terminus. Regions 104 and 108 of the anchor primer 100 contain nucleotides complementary to regions 112 and 114, respectively. Thus, sequences in regions 104 and 108 allow for capture of the termini of a linear target nucleic acid. For some applications, the terminal regions 104 and 108 can include recognition sequences for restriction enzymes, along with cDNA or genomic sequences adjacent to these recognition sequences, e.g. sequences at the termini of fragments of cDNA or genomic DNA digested with restriction enzymes. Thus, regions 104 and 108 can be designed to capture single stranded nucleic acids corresponding to restriction enzyme-generated fragments.

Annealing of the target nucleic acid 110 to the primer 100 as shown in FIG. 1 leaves the 3'-OH terminus of the target nucleic acid 110 available for either primer extension or ligation to a polynucleotide. In primer extension, the 3' hydroxyl group at the terminus of the target nucleic acid 112 is extended in the presence of a DNA polymerase, nucleotide triphosphates and any desired associated factors using the gap region 106 of anchor primer 100 as a template. Complete extension of the primer results in juxtaposition of the free 3'-OH group of the extended primer and the 5'-PO₄ of the linear target nucleic acid 110. In the presence of a ligase, the 5'PO₄ and 3'-OH can be covalently attached. This results in circularization of the target nucleic acid 110. While regions 104 and 108 of the anchor primer 100 preferably contain nucleotides perfectly complementary to regions 112 and 114, respectively, in the primer extension this requirement, some mismatch can be tolerated between the anchor primer and target nucleic acid molecules. In an alternative embodiment, the annealed target nucleic acid 110 can be circularized using a gap oligonucleotide 116 using annealing and ligation. In this embodiment, the terminal regions 112 and 114 of the target nucleic acid are preferably perfectly complementary to regions 104 and 108 of the anchor primer 100. The gap oligonucleotide 116 is complementary to nucleotides in the gap region 106 of the anchor primer and mixed with the anchor primer 106 under conditions that allow for it to anneal to the anchor primer. In the presence of ligase, the gap oligonucleotide 116 will become covalently linked by its 3 'OH group to the 5'-PO₄of the target nucleic acid. The 5'PO₄ of the gap oligonucleotide will be similarly linked to the 3'-OH of the target nucleic acid sequences. Preferably, the gap oligonucleotide 116 is perfectly complementary to the gap region 106. A highly sensitive system for detecting a nucleic acid system is therefore provided by requiring that a circular nucleic acid be produced based on (a) homology at the 5' and 3' ends of a linear target nucleic acid sequence, and (b) two separate ligation events.

If desired, an annealed circularized nucleic acid can be sequenced directly. In preferred embodiments, the 3' terminal region of the anchor primer includes a query nucleotide, e.g., the 3' terminus can be within 15, 10, 5, 4, 3, 2, 1, nucleotides of, or can be at, the 3' terminus of the anchor primer. The query nucleotide is a nucleotide able to detect a nucleotide associated with a predetermined SNP. A target nucleic acid under is annealed with the anchor primer under conditions that result in hybridization of the target nucleic acid molecule if it contains a nucleotide homologous to the query nucleotide. A successful annealing event allows for primed extension of the anchor primer using the target nucleic acid molecule as a template. Conversely, attempted annealing of a target nucleic acid sequence lacking a nucleotide complementary to the query nucleotide results in a mismatch between the query nucleotide in the anchor primer and the target nucleic acid. The anchor primer cannot be extended in this case.

For a given SNP, anchor primer probes vary in their query nucleotides according to the nucleotides associated with the SNP. Thus, for a SNP defined by the presence of an 'A' nucleotide in some members of a population, and 'C nucleotide at other positions, the corresponding query nucleotides in the anchor primer can be 'C and ', respectively. Polymerases lacking exonucleolytic activity e.g., exo^" or mutS polymerase, particularly suited using this approach. Alternatively, the circularized nucleic acid can be amplified and the sequence of the amplified products determined. The 3'-OH terminus of the anchor primer 100 is available to prime a sequencing reaction, e.g., a dideoxy sequencing reaction.

The 3'-OH terminus of the anchor primer 100 can alternatively prime amplification, e.g., rolling-circle amplification (RCA), with the circularized nucleic acid acting as the template nucleic acid. RCA using the 3'-OH of the anchor primer results in molecule covalently linked to the substrate 102 that contains multiple copies of a sequence complementary to the circularized nucleic acid template.

Another anchor primer according to the invention is shown in FIG. 2. An anchor primer 200 is attached at its 5' end to a substrate 202. The anchor primer includes two adjoining regions, 204 and 206, which are complementary to regions.

Annealed to the anchor primer 200 in FIG. 2 is a linear target nucleic acid 210 having regions 212 at its 3' end and region 214 at its 5' end. Regions 204 and 206 of the anchor primer 200 anneal to regions 212 and 214, respectively. Thus, by including sequences complementary to the termini of a restriction enzyme generated fragment in regions 204 and 206, the anchor primer 200 can capture desired restriction fragments. Suitable fragments include those containing, or suspected of containing, polymorphisms, such as single nucleotide polymorphisms.

A third type of anchor primer is shown in FIG. 3. The figure shows an anchor primer 300 attached at its 5' end to a substrate 302. The anchor primer 300 includes near its 5' end a nucleotide region 304.

Also shown in FIG.3 is a circular target nucleic acid 310. The target nucleic acid 310 includes a region 312 complementary to the nucleotide region 304.

Also present in the target nucleic acid 310 is a nucleic region 350. In preferred embodiments, the nucleotide region 304 is complementary to a nucleotide region 312 present in a plurality of target nucleic acids. In still more preferred embodiments, the nucleotide region 312 is present in a sequence, e.g., a vector, which has been ligated to members of a starting population of nucleic acids.

An anchor primer may optionally contain additional elements, e.g., spacer sequences at its 5' terminus, one or more restriction enzyme recognition sites, RNA polymerase binding sites (e.g., a T7 promoter site).

One or more of the adapter regions may include, e.g., a restriction enzyme recognition site or sequences present in identified DNA sequences, e.g., sequences present in known genes. One or more adapter regions may also include sequences that identify sequence known to flank and/or encompass sequence polymorphisms. Examples of such sequences include the query nucleotides. Sequence polymorphisms include nucleotide substitutions, insertions, deletions, or other rearrangements that result in a sequence difference between two otherwise identical nucleic acid sequences. An example of a sequence polymorphism is a single nucleotide polymorphism (SNP).

In other embodiments, the anchor primer arrays described herein can be used to identify or otherwise characterize target nucleic acid sequences that are circularized prior to being added to the arrays. Linking of Anchor Primers to a Solid Support

In general, any nucleic acid capable of base pairing can be used as an anchor primer. In some embodiments, the anchor primer is an oligonucleotide. As utilized herein the term oligonucleotide includes linear oligomers of natural or modified monomers or linkages, e.g., deoxyribonucleosides, ribonucleosides, anomeric forms thereof, peptide nucleic acids (PNAs), and the like, that are capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions. These types of interactions can include, e.g., Watson-Crick type of base-pairing, base stacking, Hoogsteen or reverse-Hoogsteen types of base-pairing, or the like. Generally, the monomers are linked by phosphodiester bonds, or analogs thereof, to form oligonucleotides ranging in size from, e.g., 3-200, 8-150, 10-100, 20- 80, or 25-50 monomeric units.

The oligonucleotides of the present invention can include non-natural nucleotide analogs. However, where, for example, processing by enzymes is required, or the like, oligonucleotides comprising naturally occurring nucleotides are generally required for maintenance of biological function. Any material can be used as the solid support material, as long as the surface allows for stable attachment of the primers and detection of nucleic acid sequences. The solid support material can be planar. In some embodiments, the solid support is optically transparent, e.g., glass.

The anchor primer can be linked to the solid support to reside on or within the solid support. The distance between anchor primers on the array will be determined in part by the method and apparatus used to detect and further analyze extended anchor primer templates. Methods for detecting extended anchor primers are discussed below. Apparatuses for detecting extended anchor primers are also discussed below and in addition include those known in the art for detecting macromolecules. PCT publication WO 00/06770, for example, discusses apparatuses such as confocal scanning microscopy, scanning near-field optical microscopy (SNOM), scanning tunneling microscopy, and atomic force microscopy. These apparatuses allow for resolution on the order of tens of manometers. Thus, in some embodiments, the plurality of anchor primers is linked to the solid support so they are spaced regular intervals within an array. The distance between primers on a solid substrate can be, e.g., 1 nm to 150μm, 10-400 μm, 50-150 μm, 100-150 μm, or 150 μm. In prefeπed embodiments, arrays are spaced at manometer resolution, e.g., 10 nm X 10 nm. Construction of arrays with manometer resolution is described in PCT application WO 00/06770.

An array of attachment sites on the optically transparent solid support is constructed using lithographic techniques commonly used in the construction of electronic integrated circuits. These techniques are described in, e.g., U.S. Patent Nos. 5,5143,854, 5,445,934, 5,744,305, and 5, 800,992; Chee et al, Science 274: 610-614 (1996); Fodor et al, Nature 364: 555-556 (1993); Fodor et al, Science 251 : 767-773 (1991); Gushin, et al, Anal Biochem. 250: 203-211 (1997); Kinosita et al, Cell 93: 21-24 (1998); Kato-Yamada et al, J. Biol Chem. 273: 19375-19377 (1998); and Yasuda et al, Cell 93: 1117-1124 (1998). Photolithography and electron beam lithography sensitize the solid support or substrate with a linking group that allows attachment of a modified biomolecule (e.g., proteins or nucleic acids). See e.g., Service, Science 283: 27-28 (1999); Rai-Choudhury, HANDBOOK OF MICROLITHOGRAPHY,

MlCROMACHINING, AND MlCROFABRiCATION, VOLUME I: MICROLITHOGRAPHY, Volume PM39, SPIE Press (1997). Alternatively, an array of sensitized sites can be generated using thin-film technology as described in Zasadzinski et al, Science 263: 1726-1733 (1994).

Additional methods for attaching nucleic acids to solid substrates use sialinized surfaces and are disclosed in US Patent No. 6,136,962. Attachment using disulfide bonds is described in US. Patent No.6,030,782. The contents of all of these patents and publications are incorporated by reference in their entirety.

Anchor primers are linked to the solid substrate at the sensitized sites. A region of a solid substrate containing a linked primer is an anchor pad. Thus, by specifying the sensitized sites on the solid support, it is possible to form an array or matrix of anchored pads. The anchor pads can, e.g., small diameter spots etched at evenly spaced intervals on the solid support. Each sensitized site on a solid support is potentially capable of attaching multiple anchor primers. Thus, each anchor pad may include one or more anchor primers. It is preferable to maximize the number of pads that have only a single productive reaction center (e.g., the number of pads that, after the extension reaction, have only a single sequence extended from the anchor primer). This can be accomplished by techniques which include, but are not limited to: (i) varying the dilution of biotinylated anchor primers that are washed over the surface; (ii) varying the incubation time that the biotinylated primers are in contact with the avidin surface; or (iii) varying the concentration of open- or closed-circular template so that, on average, only one primer on each pad is extended to generate the sequencing template. In some embodiments, each individual pad contains just one linked anchor primer.

Pads having only one anchor primer can be made by performing limiting dilutions of a selected anchor primer on to the solid support such that, on average, only one anchor primer is deposited on each pad. The concentration of anchor primer to be applied to a pad can be calculated utilizing, for example, a Poisson distribution model. In order to maximize the number of reaction pads that contain a single anchor primer, a series of dilution experiments are performed in which a range of anchor primer concentrations or circular template concentrations are varied. For highly dilute concentrations of primers, primers and circular templates binding to the same pad will be independent of each other, and a Poisson distribution will characterize the number of anchor primers extended on any one pad. Although there will be variability in the number of primers that are actually extended, a maximum of 37% of the pads will have a single extended anchor primer (the number of pads with a single anchor oligonucleotide). This number can be obtained as follows.

Let N_p be the average number of anchor primers on a pad and f be the probability that an anchor primer is extended with a circular template. Then the average number of extended anchor primers per pad is N_pf, which is defined as the quantity a. There will be variability in the number of primers that are actually extended. In the low-concentration limit, primers and circular templates binding to the same pad will be independent of each other, and a Poisson distribution P(n) will characterize the number of anchor primers n extended on any pad. This distribution may be mathematically defined by: P(n) = ( a"/ n!)exp(-a), with P(l) = a exp(-a). The probability P(l) assumes it maximum value exp(-l) for a = 1 , with 37% of pads having a single extended anchor primer.

A range of anchor primer concentrations and circular template concentrations may be subsequently scanned to find a value of N_pf closest to 1. A preferable method to optimize this distribution is to allow multiple anchor primers on each reaction pad, but use a limiting dilution of circular template so that, on average, only one primer on each pad is extended to generate the sequencing template.

Alternatively, at low concentrations of anchor primers,, at most one anchor primer will likely be bound on each reaction pad. A high concentration of circular template may be used so that each primer is likely to be extended.

Where the reaction pads are arrayed on a planar surface or a fiber optic array. (FORA), the individual pads are approximately 10 μm on a side, with a 100 μm spacing between adjacent pads. Hence, on a 1 cm surface a total of approximately 10,000 microreactors could be deposited, and, according to the Poisson distribution, approximately 3700 of these will contain a single anchor primer. In certain embodiments, after the primer oligonucleotide has been attached to the solid support, modified, e.g., biotinylated, enzymes are deposited to bind to the remaining, unused avidin binding sites on the surface.

In other embodiments multiple anchor primers are attached to any one individual pad in an array. Limiting dilutions of a plurality of circular nucleic acid templates (described in more detail below) may be hybridized to the anchor primers so immobilized such that, on average, only one primer on each pad is hybridized to a nucleic acid template. Library concentrations to be used may be calculated utilizing, for example, limiting dilutions and a Poisson distribution model. The anchor primer can be attached to the solid support via a covalent or non-covalent interaction. Examples of such linkages common in the art include Ni²7hexahistidine, streptavidin/biotin, avidin/biotin, glutathione S-transferase (GST)/glutathione, monoclonal antibody/antigen, and maltose binding protein/maltose. Samples containing the appropriate tag are incubated with the sensitized substrate so that a single molecule attaches at each sensitized site.

The biotin-(strept-)avidin methodology provides several different ways to immobilize the anchor on the solid support. One biotin-(strept-)avidin-based anchoring method uses a thin layer of a photoactivatable biotin analog dried onto a solid surface. (Hengsakul and Cass, 1996. Biocongjugate Chem. 7: 249-254) . The biotin analog is then exposed to white light through a mask, so as to create defined areas of activated biotin. Avidin (or streptavidin) is then added and allowed to bind to the activated biotin. The avidin possesses free biotin binding sites that can be utilized to "anchor" the biotinylated oligonucleotides through a biotin-(strept-)avidin linkage. Alternatively, the anchor primer can be attached to the solid support with a biotin derivative possessing a photo-removable protecting group. This moiety is covalently bound to bovine serum albumin (BSA), which is attached to the solid support, e.g., a glass surface. See Pirrung and Huang, 1996. Bioconjugate Chem. 7: 317-321. A mask is then used to create activated biotin within the defined iπadiated areas. Avidin may then be localized to the irradiated area, with biotinylated DNA subsequently attached through a BSA-biotin-avidin- biotin link. If desired, an intermediate layer of silane is deposited in a self-assembled monolayer on a silicon dioxide silane surface that can be patterned to localize BSA binding in defined regions. See e.g., Mooney, ET al, 1996. Proc. Natl. Acad. Sci. USA 93: 12287-12291. Each sensitized site on a solid support is potentially capable of attaching multiple anchor primers. Thus, each anchor pad may include one or more anchor primers. It is preferable to maximize the number of pads that have only a single productive reaction center (e.g., the number of pads that, after the extension reaction, have only a single sequence extended from the anchor primer). This can be accomplished by, e.g. : (i) varying the dilution of biotinylated anchor primers that are washed over the surface; (ii) varying the incubation time that the biotinylated primers are in contact with the avidin surface; or (iii) varying the concentration of open- or closed-circular template to maximize the number of pads on which only primer is extended to generate the sequencing template. Preparing template nucleic acids The template nucleic acids can in general be derived from any source, as long as at least a portion of the template nucleic acids are linear and single-stranded when annealed to the anchor primer arrays. Preferably a template nucleic acid containing a target nucleic acid is in the form an open circle. An "open circle" is a linear single-stranded nucleic acid molecule having a 5' phosphate group and a 3' hydroxyl group, i.e., the ends of a given open circle nucleic acid molecule can be ligated by DNA ligase. Open circles are described in detail in Lizardi, U.S. Pat. No. 5, 854,033. An open circle can be converted to a closed circle in the presence of a DNA ligase (for DNA) or RNA ligase following, e.g., annealing of the open circle to an anchor primer.

In preferred embodiments, at least one linear target nucleic acid in a population of nucleic acids will have sequences at its 5' and 3' ends, i.e., regions 112 and 114 as shown in FIG. 1, that are complementary to regions 104 and 108 of an anchor primer.

The 5'- and 3'-terminal regions 112 and 114 of the oligonucleotides are designed to basepair adjacent to one another on a specific target sequence strand, thus the termini of the linear oligonucleotide are brought into juxtaposition by hybridization to the target sequence. This juxtaposition allows the two probe segments (if properly hybridized) to be covalently- bound by enzymatic ligation (e.g., with T₄ DNA ligase), thus converting the probes to circularly-closed molecules which are catenated to the specific target sequences (see e.g., Nilsson, et al, 1994. Science 265: 2085-2088). The resulting probes are suitable for the simultaneous analysis of many gene sequences (see e.g., Lizardi, et al, 1998. Nat. Genet. 19: 225-232; Nilsson, et al, 1997. Nat. Genet. 16: 252-255).

The starting population of nucleic acid can be either single-stranded or double- stranded, as long as it includes a region that, if present in the library, is available for annealing, or can be made available for annealing, to an anchor primer sequence.

Library templates can include multiple elements, including, but not limited to, one or more regions that are complementary to the anchor primer. For example, the template libraries may include a region complementary to a sequencing primer, a control nucleotide region, and an insert sequence containing a sequence of interest, i.e., the sequencing template to be subsequently characterized. As utilized herein the term "complement" refers to nucleotide sequences that are able to hybridize to a specific nucleotide sequence to form a matched duplex.

In one embodiment, a library template includes: (/^') two distinct regions, e.g., regions 114 and 112, that are complementary to the anchor primer, (//) one region complementary to a sequencing primer, (iii) one control nucleotide region, (iv) an insert sequence of 30 - 100 nucleotides that is to be sequenced. The template can, of course, include two, three, or all four of these features.

The template nucleic acid can be constructed from any source of nucleic acid, e.g., any cell, tissue, or organism, and can be generated by any art-recognized method. Suitable methods include, e.g., sonication of genomic DΝA and digestion with one or more restriction endonucleases (RE) to fragment a population of nuclei acid molecules, e.g., genomic DΝA. The restriction enzymes can recognize eight or six base recognition sequences, or can recognize degenerate sequences. In some embodiments, the restriction enzymes are Type II or Type IIS restriction enzymes. Preferably, one or more of the restriction enzymes have distinct four-base recognition sequences. Examples of such enzymes include, e.g., Sau3Al, Mspl, and Taql. Preferably, the enzymes are used in conjunction with anchor primers having regions containing recognition sequences for the corresponding restriction enzymes. In some embodiments, the one or both adapter regions anchor primers contain additional sequences adjoining known restriction enzyme recognition sequences, thereby allowing for capture or annealing of specific restriction fragments of interest to the anchor primer.

The target nucleic acid can also be generated by amplification, e.g., primed amplification of a desired target sequence. Alternatively, template libraries can be made by generating a complementary DNA

(cDNA) library from RNA, e.g., messenger RNA (mRNA). The cDNA library can, if desired, be further processed with restriction endonucleases and/or primed amplification to obtain either 3' signature sequences, internal fragments, or 5' fragments. The libraries can be alternatively be constructed in vectors having sequences complementary to adapter regions in an anchor primer. In some embodiments, the libraries contain a sequence of interest, e.g., a known or suspected sequence polymorphism on a restriction fragment.

Annealing and Amplification of Primer-Template Nucleic Acid Complexes

Libraries of nucleic acids are annealed to anchor primer sequences using recognized techniques (see, e.g., Hatch, et al, 1999. Genet. Anal Biomol. Engineer. 15: 35-40; Kool, U.S. Patent No. 5,714, 320 and Lizardi, U.S. Patent No. 5,854,033). In general, any procedure for annealing the anchor primers to the template nucleic acid sequences is suitable as long as it results in formation of specific, i.e., perfect or nearly perfect, complementarity between the adapter region or regions in the anchor primer sequence and a sequence present in the template library.

In some embodiments, the circularized template is amplified. A number of in vitro nucleic acid amplification techniques may be utilized to extend the anchor primer sequence. Preferably, the size of the amplified DNA should be smaller than the size of the anchor pad and also smaller than the distance between anchoring pads. The amplification is typically performed in the presence of a polymerase, e.g., a DNA or RNA-directed DNA polymerase, and one, two, three, or four types of nucleotide triphosphates, and, optionally, auxiliary binding proteins. In general, any polymerase capable of extending a primed 3' -OH group can be used a long as it lacks a 3' to 5' exonuclease activity. Suitable polymerases include, e.g., the DNA polymerases from Bacillus stearothermophilus, Thermus acquaticus, Pyrococcus furiosis, Thermococcus litoralis, and Thermus thermophilus, bacteriophage T₄ and T₇, and the E. coli DNA polymerase I Klenow fragment. Suitable RNA-directed DNA polymerases include, e.g., the reverse transcriptase from the Avian Myeloblastosis Virus, the reverse transcriptase from the Moloney Murine Leukemia Virus, and the reverse transcriptase from the Human Immunodeficiency Virus-I.

A number of in vitro nucleic acid amplification techniques have been described. These amplification methodologies may be differentiated into those methods: (i) which require temperature cycling - polymerase chain reaction (PCR) (see e.g., Saiki, et al, 1995. Science 230: 1350-1354), ligase chain reaction (see e.g., Barany, 1991. Proc. Natl. Acad. Sci. USA 88: 189-193; Barringer, et al, 1990. Gene 89: 1 17-122) and transcription-based amplification (see e.g., Kwoh, et al, 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177) and (ii) isothermal amplification systems - self-sustaining, sequence replication (see e.g., Guatelli, et al, 1990. Proc. Natl Acad. Sci. USA 87: 1874-1878); the Qβ replicase system (see e.g., Lizardi, et al, 1988. BioTechnology 6: 1197-1202); strand displacement amplification Nucleic Acids Res. 1992 Apr l l;20(7):1691-6.; and the methods described in PNAS 1992 Jan l ;89(l):392-6; and NASBA J Virol Methods. 1991 Dec;35(3):273-86.

Isothermal amplification also includes rolling circle-based amplification (RCA). RCA is discussed in, e.g., Kool, U.S. Patent No. 5,714,320 and Lizardi, U.S. Patent No. 5,854,033; Hatch, et al, 1999. Genet. Anal. Biomol Engineer. 15: 35-40. The result of the RCA is a single DNA strand extended from the 3' terminus of the anchor primer (and thus is linked to the solid support matrix) and including a concatamer containing multiple copies of the circular template annealed to a primer sequence. Typically, 10,000 or more copies of circular templates, each having a size of approximately 100 nucleotides size range, can be obtained with RCA.

The product of RCA amplification following annealing of a circular nucleic acid molecule to an anchor primer is shown schematically in FIG. 4. A circular template nucleic acid 402 is annealed to an anchor primer 404, which has been linked to a surface 106 at its 5' end and has a free 3' OH available for extension. The circular template nucleic acid 402 includes two adapter regions 408 and 410 that are complementary to regions of sequence in the anchor primer 404. Also included in the circular template nucleic acid 402 is an insert 412 and a region 414 complementary to a sequencing primer, which is used in the sequencing reactions described below. Upon annealing, the free 3'-OH on the anchor primer 404 can be extended using sequences within the template nucleic acid 402. The anchor primer 402 can be extended along the template multiple times , with each iteration adding to the sequence extended from the anchor primer a sequence complementary to the circular template nucleic acid. Four iterations, or four rounds of rolling circle amplification, are shown in FIG.4 as the extended anchor primer amplification product 414. Extension of the anchor primer results in an amplification product covalently attached to the substrate 406.

RCA-based amplification has recently been utilized to obtain an isothermal cascade amplification reaction of circularized padlock probes in vitro in order to detect single-copy genes in human genomic DNA samples (see Lizardi, et al, 1998. Nat. Genet. 19: 225-232). In addition, RCA has also been utilized to detect single DNA molecules in a solid phase-based assay (see Lizardi, et al, 1998. Nat. Genet. 19: 225-232).

Rolling-circle amplification (RCA) can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions. In the presence of two primers (one hybridizing to the + strand, and the other, to the - strand of DNA), concatamers of both strands can be simultaneously synthesized. This allows for lxlO'or more copies of each circle in a short period of time (i.e., less-than 90 minutes), enabling the detection of single- point mutations within the human genome. Using a single primer, RCA generates hundreds of randomly linked copies of a covalently closed circle in several minutes. If solid support matrix-associated, the DNA product remains bound at the site of synthesis, where it may be labeled, condensed, and imaged as a point light source. For example, linear oligonucleotide probes, which can generate RCA signals, have been bound covalently onto a glass surface. The color of the signal generated by these probes indicates the allele status of the target, depending upon the outcome of specific, target-directed ligation events. As RCA permits millions of individual probe molecules to be counted and sorted, it is particularly amenable for the analysis of rare somatic mutations. RCA also shows promise for the detection of padlock probes bound to single-copy genes in cytological preparations. In addition, solid-phase RCA methods have also been developed to provide an effective method of detecting constituents within a solution. Initially, a recognition step is used to generate a complex consisting of a DNA primer duplexed with a circular template is bound to a surface. A polymerase enzyme is then used to amplify the bound complex. RCA uses small DNA probes that are amplified to provide an intense signal using detection methods, including the methods described in more detail below.

Other examples of isothermal amplification systems include, e.g., (i) self-sustaining, sequence replication (see e.g., Guatelli, et al, 1990. Proc. Natl. Acad. Sci. USA 87: 1874- 1878), (ii) the Qβ replicase system (see e.g., Lizardi, et al, 1988. BioTechnology 6: 1197- 1202), and (iii) nucleic acid sequence-based amplification (NASBA™; see Kievits, et al, 1991.

J. Virol Methods 35: 273-286).

Methods of identifying extended anchor primers

In general, the oligonucleotide aπays described herein can be used in conjunction with any substrate-based sequencing method known in the art. The methods can also be used in association with conventional optic, as well as fiber-optic based detection signal detection methods. The extended anchor primers produced using the arrays and methods disclosed herein can be further analyzed using methods known in the art. For example, the sequence of an extended anchor primer can be determined, or sequences homologous to a probe molecule can be identified. Some of these methods are described below.

One or more sequences in the extended anchor primer can be determined by subjecting the extended anchor primer product to dideoxy terminator sequencing. Analysis of the sequencing products allows the identification of one or more nucleotides in the extended anchor primer. Alternatively, pyrophosphate-based sequencing methods can be used to identify nucleotides in an extended anchor primer. For example, Nyren et al. (Analytical Biochemistry 208:171-175, 1993) have described a method relying on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA) These methods can be used in as part of microsequencing assays. These assays are based on extension of a single nucleotide from a sequencing primer that hybridizes just upstream of a polymorphic base of interest in the extended anchor primer. A polymerase is used to specifically extend the 3' end of the primer with one single ddNTP (chain terminator) complementary to the selected nucleotide at the polymorphic site. Next, the identity of the incorporated nucleotide is determined. In one embodiment, microsequencing reactions are carried out using fluorescent ddNTPs, and the extended microsequencing primers are analyzed to determine the identity of the incorporated nucleotide. Microsequencing reactions are described in EP 412 883. Incorporated ddNTPs can alternatively be radiolabeled (Syvάnen, Clinica Chimica Acta 226:225-236, 1994) or linked to fluorescein (Livak and Hainer, Human Mutation 3:379- 385,1994). The detection of radiolabeled ddNTPs can be achieved through scintillation-based techniques. The detection of fluorescein-linked ddNTPs can be based on the binding of antifluorescein antibody conjugated with alkaline phosphatase, followed by incubation with a chromogenic substrate (such as ?-nitrophenyl phosphate). Other possible reporter-detection pairs include, e.g., ddNTP linked to dinitrophenyl (DNP) and anti-DNP alkaline phosphatase conjugate (Harju et al., Clin. Chem. 39/11 2282-2287, 1993) or biotinylated ddNTP and horseradish peroxidase-conjugated streptavidin with o-phenylenediamine as a substrate (WO 92/15712). Other approaches recognized in the art can be used to detect the nucleotide added to the microsequencing primer. For example, homogeneous phase detection method based on fluorescence resonance energy transfer has been described by Chen et al., Nucleic Acids Research 25:347-353 1997) and Chen et al., Proc. Natl. Acad. Sci USA 94/20 10756- 10761,1997). Amplified genomic DNA fragments containing polymorphic sites are incubated with a 5'-fluorescein-labeled primer in the presence of allelic dye-labeled dideoxyribonucleoside triphosphates and a modified Taq polymerase. The dye-labeled primer is extended one base by the dye-terminator specific for the allele present on the template. At the end of the genotyping reaction, the fluorescence intensities of the two dyes in the reaction mixture are analyzed directly without separation or purification. All these steps can be performed in the same tube and the fluorescence changes can be monitored in real time. Alternatively, the extended primer may be analyzed by MALDI-TOF Mass Spectrometry. The base at the polymorphic site is identified by the mass added onto the microsequencing primer (see Haff L.A. and Smirnov I. P., Genome Research, 7:378-388, 1997). The extended anchor primers can also be detected using mismatch detection assays based on polymerases and/or ligases in enzyme based mismatch detection assays. The terms "enzyme based mismatch detection assay" are used herein to refer to any method of determining the allele of a biallelic marker based on the specificity of ligases and polymerases. An example of these methods is the oligonucleotide ligation assay ("OLA"). OLA uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target molecules. One of the oligonucleotides is biotinylated, and the other is detectably labeled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate that can be captured and detected. OLA is described in Nickerson D.A. et al. (Proc. Natl. Acad. Sci. US.A. 87:8923-8927, 1990). In preferred embodiments, an extra amplification step, such as PCR, is used to achieve the exponential amplification of target DNA, which is then detected using OLA. A second ligation-based method is ligase chain reaction ("LCR"). The sequences of each pair of oligonucleotides in LCR is selected to permit the pair to hybridize to abutting sequences of the same strand of the target. Such hybridization forms a substrate for a template-dependent ligase. LCR can be performed on extended anchor primers with oligonucleotides having the proximal and distal sequences of the same strand of an extended anchor primer. In one embodiment, either oligonucleotide will be designed to include a site of interest on the extended anchor primer. In such an embodiment, the reaction conditions are selected such that the oligonucleotides can be ligated together only if the target molecule either contains or lacks the specific nucleotide(s) that is complementary to the site of interest on the oligonucleotide. In an alternative embodiment, the oligonucleotides will not include the biallelic marker, such that when they hybridize to the target molecule, a "gap" is created as described in WO 90/0 1069. This gap is then "filled" with complementary dNTPs (as mediated by DNA polymerase), or by an additional pair of oligonucleotides. Thus at the end of each cycle, each single strand has a complement capable of serving as a target during the next cycle and exponential allele-specific amplification of the desired sequence is obtained.

Ligase/Polymerase-mediated Genetic Bit Analysis™ is another method for determining the identity of a nucleotide at a preselected site in a nucleic acid molecule (WO 95/21271). This method includes incorporation of a nucleoside triphosphate that is complementary to a nucleotide present at the preselected site onto the terminus of a primer molecule, and subsequent ligation of the primer to a second oligonucleotide. The reaction is monitored by detecting a specific label attached to the reaction's solid phase or by detection in solution.

A further method of determining the identity of an extended anchor primer involves nucleic acid hybridization. Specific probes can be designed that hybridize preferentially or specifically to one form of an extended anchor primer. Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alternative sequences of an extended anchor primer. For example, conditions are preferably chosen to discriminate between hybridization of a probe sequence to a perfectly matched homologous extended anchor primer sequence and an extended anchor primer sequence containing one or more mismatches with the probe sequence. Stringent, sequence specific hybridization conditions, under which a probe will hybridize only to the exactly complementary target sequence are well known in the art (see, e.g., Sambrook et al., Molecular Cloning — A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., 1989). Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. By way of example and not limitation, procedures using conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65° C in buffer composed of 6X SSC, 50 mM Tris-HC 1 (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65°C, the preferred hybridization temperature, in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20 X 10⁶ cpm of ³²P-labeled probe. Alternatively, the hybridization step can be performed at 65°C in the presence of SSC buffer, 1 x SSC corresponding to 0.15M NaCl and 0.05 M Na citrate. Subsequently, filter washes can be done at 37°C for 1 h in a solution containing 2X SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by a wash in 0.1X SSC at 50°C for 45 min. Alternatively, filter washes can be performed in a solution containing 2 x SSC and 0.1% SDS, or 0.5 x SSC and 0.1% SDS, or 0.1 x SSC and 0.1% SDS at 68°C for 15 minute intervals. Following the wash steps, the hybridized probes are detectable by autoradiography.

Alternatively, in some embodiments, extended anchor primers containing a limited number of mismatches to a target sequence can be detected with a probe sequence. Such extended anchor primers can be detected using conditions of intermediate stringency. By way of example and not limitation, procedures using conditions of intermediate stringency are as follows: Filters containing DNA are prehybridized, and then hybridized at a temperature of 60°C in the presence of a 5 x SSC buffer and labeled probe. Subsequently, filters washes are performed in a solution containing 2x SSC at 50°C and the hybridized probes are detectable by autoradiography.

Highly sensitive hybridization assays with no need for separations or washes have been described (see Landegren et at., Genome Research, 8:769-776,1998). For example, the TaqMan™ assay takes advantage of the 5' nuclease activity of Taq DNA polymerase to digest a DNA probe annealed specifically to the accumulating amplification product. TaqMan™ probes are labeled with a donor-acceptor dye pair that interacts via fluorescence energy transfer. Cleavage of the TaqMan™ probe by the advancing polymerase during amplification dissociates the donor dye from the quenching acceptor dye, greatly increasing the donor fluorescence. All reagents necessary to detect two allelic variants can be assembled at the beginning of the reaction and the results are monitored in real time (see Livak et al., Nature Genetics, 9:341-342, 1995). In an alternative homogeneous hybridization-based procedure, molecular beacons are used for allele discriminations. Molecular beacons are hairpin-shaped oligonucleotide probes that report the presence of specific nucleic acids in homogeneous solutions. When they bind to their targets they undergo a conformational reorganization that restores the fluorescence of an internally quenched fluorophore (Tyagi et al., Nature Biotechnology, 16:49-53, 1998).

OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. An array of oligonucleotide anchor primers, the array comprising: a support having a first surface; and a plurality of different anchor primers attached to the surface of the support, wherein at least one of said anchor primers in said plurality comprises: a first region at the 3' end of said anchor primer, said first region complementary to a 5' region of a target nucleic acid sequence; and a second region at the 5' end of said anchor primer, said second region complementary to a 3' region of said target nucleic acid sequence; and optionally, a third region between the first region and second region of said anchor primer; wherein said anchor primer is attached to the surface of the solid support at a predetermined region of said surface.

2. The aπay of claim 1, wherein at least one of said anchor primers is covalently linked to at least one copy of a sequence complementary to said target nucleic acid sequence.

3. The aπay of claim 1, wherein at least one of said anchor primers includes a first region comprising a query nucleotide complementary to a nucleotide defining a sequence polymorphism in a target nucleic acid.

4. The array of claim 1, wherein said query nucleotide is at the 3' terminus of said oligonucleotide.

5. The array of claim 1, wherein each anchor primer is 12 to 100 nucleotides in length.

6. The array of claim 1, wherein each anchor primer is 24 to 40 nucleotides in length.

7. The array of claim 1, wherein the array comprises at least 100 different oligonucleotides attached to a solid support.

8. The aπay of claim 1, wherein the aπay comprises at least 10,000 different oligonucleotides attached to a solid support.

9. The aπay of claim 1, wherein the array comprises at least 100,000 different oligonucleotides attached to a solid support.

10. The array of claim 1, wherein each of the predefined regions is physically separated from each of the other predetermined regions.

11. The array of claim 1, wherein at least two of the predefined regions are separated by 10-400 μm.

12. The aπay of claim 1, wherein at least two of the predefined regions are separated by 50-150 μm.

13. The aπay of claim 1, wherein the length of the third region of said anchor primers region is about 4 to 20 nucleotides.

14. The array of claim 13, wherein the length of the third region of said anchor primers is about 6 to 18 nucleotides.

15. The aπay of claim 13, wherein the length of the third regions of said anchor primers is about 8 to 16 nucleotides.

16. A method of determining the sequence of a nucleic acid, the method comprising: providing the aπay of claim 1; contacting the aπay with an open or closed circular target nucleic acid under conditions which allow the target nucleic acid to anneal to one or more anchor primers in the aπay; and determining the nucleotide sequence of at least a portion of the target nucleic acid.

17. The method of claim 16, wherein the anchor primers in said aπay comprise a second region.

18. The method of claim 17, wherein said circular target nucleic acid is formed by: annealing an oligonucleotide in the aπay with a linear target nucleic acid having 5' and

3' termini under conditions which allow the 5' and 3' teπnini of the target nucleic acid to anneal to complementary sequences in an anchor primer in said aπay; contacting the annealed teπnini of the target nucleic acid with a gap oligonucleotide complementary to the second region of gap region of at least one of said anchor primers and a ligase under conditions sufficient allow for ligation of the 5' and 3' termini of the target nucleic acid to the gap oligonucleotide, thereby forming a closed circular target nucleic acid.

19. The method of claim 16, wherein said covalently closed circular target nucleic acid is amplified prior to determining its nucleotide sequence.

20. The method of claim 19, wherein said amplifying comprises: extending the annealed anchor primer with a polymerase under conditions which allow for formation of a polynucleotide product complementary to one or more copies of said covalently closed circular target nucleic acid; and identifying the polynucleotide product, thereby determining the sequence of the circularized target nucleic acid.

21. The method of claim 16, wherein said target nucleic acid contains a sequence polymorphism.

22. The method of claim 21, wherein the single nucleotide polymorphism is complementary to a query nucleotide in said first region of said anchor primer.

23. The method of claim 22, wherein said query nucleotide is at the 3' terminus of said anchor primer.

24. The method of claim 23, wherein said covalently closed circular nucleic acid is formed by: contacting the substrate with a linear target nucleic acid under conditions to allow the 5' and 3' termini of the target nucleic acid to anneal to said anchor primers, wherein said 5' and 3' termini separated by a gap region of at least 6 nucleotides on said anchor primer; extending the 3' terminus of said target nucleic acid with a polymerase under condition sufficient to form an extended product whose 3' terminal nucleotide abuts the 5' terminus of the annealed target nucleic acid; and ligating the 3' terminal nucleotide of the extended product to the 5' terminus of the annealed target nucleic acid, thereby forming a closed circular molecule.

25. The method of claim 16, wherein said target nucleic acid is derived from a population of RNA molecules.

26. The method of claim 16, wherein said target nucleic acid is derived from a population of DNA molecules.

27. A method for comparing the relative amount of a target nucleic acid in a first and second population of nucleic acid molecules, the method comprising: providing one or more or more nucleic acid anchor primers linked to a solid support; providing a first plurality of circular single-stranded nucleic acid templates derived from the first population of nucleic acid molecules and a second plurality of circular single- stranded nucleic acid molecules derived from the second population of nucleic acid molecules; annealing an effective amount of a nucleic acid anchor primer to at least one of the single-stranded circular templates derived from the first plurality of circular single-stranded nucleic acid molecules, thereby yielding a first primed single-stranded circular template; annealing an effective amount of the nucleic acid anchor primers to at least one of the single-stranded circular templates from the second plurality of circular single-stranded nucleic acid molecules, thereby yielding a second primed single-stranded circular template; combining the first and second primed anchor primer-circular template complexes with a polymerase to generate multiple copies of the circular nucleic acid template derived from the first and second populations of nucleic acid molecules; annealing an effective amount of a sequencing primer to the first and second circular nucleic acid templates to yield first and second primed sequencing primer-circular nucleic acid template complexes; extending the sequencing primers on the first and second primed sequencing primer- circular nucleic acid template complexes with a polymerase and a predetermined nucleotide triphosphate to yield a first and second sequencing product associated with the first and second primed sequencing primer-circular nucleic acid template complexes; measuring the levels of the first and second sequencing products, wherein the relative level of the first and second sequencing products indicates the relative level of the target nucleic acid in the first and second population of nucleic acid sequences.

28. The method of claim 27, wherein the first and second population of nucleic acid molecules are genomic DNA sequences.

29. The method of claim 27, wherein the first and second population of nucleic acid sequences are derived from RNA sequences.

30. The method of claim 27, wherein the first population of nucleic acids is from a cell population that has been exposed to a stimulus and the second population of nucleic acids is from the same cell population that has not been exposed to the stimulus.

31. The method of claim 30, wherein the stimulus is a physical stimulus.

32. The method of claim 30, wherein the stimulus is a mitogen.

33. The method of claim 30, wherein the stimulus is a ligand for a receptor expressed by cells in the cell population.

34. The method of claim 29, wherein the first and second population of nucleic acids are obtained from cells of two different individuals.

35. The method of claim 29, further comprising the relative amount of the target nucleic acid in the first and second populations with the amount of the target nucleic acid in a third population of nucleic acid molecules.

36. A method of generating a nucleic acid concatamer covalently linked to a surface, the method comprising: annealing said oligonucleotide anchor primer a linear target nucleic acid having 5' and 3' termini under conditions which allow the 5' and 3' termini of the target nucleic acid to anneal to said first and second regions in said anchor primer; contacting the annealed termini of the target nucleic acid with a gap oligonucleotide complementary to the second region of said anchor primers and a ligase under conditions sufficient allow for ligation of the 5' and 3' termini of the target nucleic acid to the gap oligonucleotide, thereby forming a closed circular target nucleic acid annealed to said anchor primer; and extending the annealed anchor primer with a polymerase under conditions which allow for formation of a polynucleotide product complementary to one or more copies of said covalently closed circular target nucleic acid, thereby generating a nucleic acid concatamer covalently linked to a surface.

37. An aπay of oligonucleotides, the aπay comprising a support having a first surface; at least one anchor primer attached to a predetermined location on said surface, said anchor primer comprising a first region complementary to a 5' region of a target nucleic acid sequence, said first region covalently linked to two or more copies of a sequence complementary to said target nucleic acid sequence; a second region at the 5' end of said anchor primer, said second region complementary to a 3' region of said target nucleic acid sequence; and optionally, a third region between the first region and second region of said oligonucleotide anchor primers, wherein said anchor primer is disposed on said support in an addressable manner.

38. The aπay of claim 37, wherein said anchor primer is attached to said support at a predetermined location on said support.

39. The use of an oligonucleotide for sequencing a nucleic acid molecule, wherein said oligonucleotide comprises: a first region at the 3' end of said oligonucleotide, said first region complementary to a 5' region of a target nucleic acid sequence; and a second region at the 5' end of said oligonucleotide, said second region complementary to a 3' region of said target nucleic acid sequence; and optionally, a third region between the first region and second region of said oligonucleotide.

40. The use according to claim 39, wherein said oligonucleotide is attached to a surface of the solid support at a predetermined region of said surface.

41. The use according to claim 40, wherein said oligonucleotide is provided as an aπay of a plurality of oligonucleotides attached at predetermined locations of said surface.

42. The use according to claim 41, wherein the aπay comprises at least 100 different oligonucleotides attached to a solid support.

43. The use according to claim 41, wherein the aπay comprises at least 10,000 different oligonucleotides attached to a solid support.

44. The use according to claim 41, wherein the aπay comprises at least 100,000 different oligonucleotides attached to a solid support.

45. The use according to claim 41, wherein each of the predefined regions is physically separated from each of the other predetermined regions.

46. The use according to claim 41, wherein at least two of the predefined regions are separated by 10-400 μm.

47. The use according to claim 41, wherein at least two of the predefined regions are separated by 50-150 μm.

48. The use according to claim 39, wherein said oligonucleotide includes a first region comprising a query nucleotide complementary to a nucleotide defining a sequence polymorphism in a target nucleic acid.

49. The use according to claim 48, wherein said query nucleotide is at the 3' terminus of said oligonucleotide.

50. The use according to claim 39, wherein said oligonucleotide is 12 to 100 nucleotides in length.

51. The use according to claim 39, wherein said oligonucleotide is 24 to 40 nucleotides in length.

52. The use according to claim 39, wherein the length of the third region of said oligonucleotide is about 4 to 20 nucleotides.

53. The use according to claim 39, further comprising annealing said oligonucleotide to a linear target nucleic acid having 5' and 3' termini under conditions which allow the 5' and 3' termini of the target nucleic acid to anneal to said first and second regions in said oligonucleotide; contacting the annealed termini of the target nucleic acid with a gap oligonucleotide complementary to the second region of said oligonucleotide and a ligase under conditions sufficient allow for ligation of the 5' and 3' termini of the target nucleic acid to the gap oligonucleotide, thereby forming a closed circular target nucleic acid annealed to said anchor primer.

54. The use according to claim 53, further comprising extending the annealed oligonucleotide with a polymerase under conditions which allow for formation of a polynucleotide product complementary to one or more copies of said covalently closed circular target nucleic acid.

55. A method of determining the sequence of a nucleic acid, the method comprising: providing an aπay comprising: a support having a first surface; a plurality of different anchor primers attached to the surface of the support, wherein at least one of said anchor primers in said plurality comprises: a first region at the 3' end of said anchor primer, said first region complementary to a 5' region of a target nucleic acid sequence; and a second region at the 5' end of said anchor primer, said second region complementary to a 3' region of said target nucleic acid sequence; and a third region between the first region and second region of said anchor primer, wherein said anchor primer is attached to the surface of the solid support at a predetermined region of said surface; contacting the aπay with a target nucleic acid under conditions which allow the target nucleic acid to anneal to one or more anchor primers in the aπay, said target nucleic acid having a 5' terminus which annals to the first region of said one or more anchor primers and a 3' terminus which anneals to the second region of said anchor primer; contacting the annealed termini of the target nucleic acid with a gap oligonucleotide complementary to the second region of gap region of at least one of said anchor primers and a ligase under conditions sufficient allow for ligation of the 5' and 3' termini of the target nucleic acid to the gap oligonucleotide, thereby forming a closed circular target nucleic acid; extending the annealed anchor primer with a polymerase under conditions which allow for formation of a polynucleotide product complementary to one or more copies of said covalently closed circular target nucleic acid; and determining the nucleotide sequence of at least a portion of the polynucleotide product.