CA2122203C - Decontamination of nucleic acid amplification reactions - Google Patents

Decontamination of nucleic acid amplification reactions Download PDF

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Publication number
CA2122203C
CA2122203C CA002122203A CA2122203A CA2122203C CA 2122203 C CA2122203 C CA 2122203C CA 002122203 A CA002122203 A CA 002122203A CA 2122203 A CA2122203 A CA 2122203A CA 2122203 C CA2122203 C CA 2122203C
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Prior art keywords
udg
amplification
sample
subsequent
prior
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Expired - Fee Related
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CA002122203A
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French (fr)
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CA2122203A1 (en
Inventor
Melinda S. Fraiser
George T. Walker
James L. Schram
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Becton Dickinson and Co
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Becton Dickinson and Co
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Publication of CA2122203A1 publication Critical patent/CA2122203A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

Abstract

Methods for inactivating contaminating amplicons in isothermal nucleic acid amplification reactions such as SDA, Q.beta. and 3SR. dU is incorporated into the amplicons produced by amplification in the place of thymine (T). If these amplicons contaminate a subsequent amplification reaction, they may be inactivated as templates (i.e., rendered unamplifiable) by treatment with UDG. As isothermal amplification does not involve elevated temperatures, the UDG may be inactivated during the subsequent amplification of specific target sequences by inclusion of the UDG inhibitor protein Ugi. Incorporation of dU has unexpectedly been found to enhance the amplification power of SDA as compared to conventional SDA
reactions. The methods may also be used to detect UDG activity in reagents or samples.

Claims (9)

1. A method for preventing amplification of contaminating amplicons generated in a prior isothermal amplification reaction during subsequent isothermal amplification of a sample, the method comprising the steps of:
a) incorporating uracil into the contaminating amplicons during the prior isothermal amplification reaction;
b) prior to the subsequent isothermal amplification, treating the sample with a sufficient amount of uracil DNA glycosylase (UDG) to render the contaminating amplicons unamplifiable, and;
c) amplifying the treated sample in the presence of a sufficient amount of uracil-DNA
glycosylase inhibitor (Ugi) to inactivate the UDG.
2. The method according to Claim 1 wherein the prior and subsequent amplifications are by Strand Displacement Amplification.
3. The method according to Claim 2 wherein 0.1-1 mM deoxy-uridinetriphosphate (dUTP) is included in the prior amplification reaction.
4. The method according to Claim 3 wherein 0.5 mM dUTP is included in the prior amplification reaction.
5. The method according to Claim 3 wherein 0.1-1 mM dUTP is included in the subsequent amplification reaction.
6. The method according to Claim 5 wherein 0.5 mM dUTP is included in the subsequent amplification reaction.
7. The method according to Claim 2 wherein the sample is treated with 0.1-10 units of UDG and the treated sample is amplified in the presence of about 0.1-50 units of Ugi.
8, The method according to Claim 7 wherein the sample is treated with 1-2 units of UDG and the treated sample is amplified in the presence of 1-4 units of Ugi.
9. The method according to Claim 2 wherein uracil is incorporated into an amplicon amplified from a target sequence of Mycobacterium tuberculosis.

19~
CA002122203A 1993-05-11 1994-04-26 Decontamination of nucleic acid amplification reactions Expired - Fee Related CA2122203C (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US6084293A 1993-05-11 1993-05-11
US08/060,842 1993-05-11

Publications (2)

Publication Number Publication Date
CA2122203A1 CA2122203A1 (en) 1994-11-12
CA2122203C true CA2122203C (en) 2001-12-18

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CA002122203A Expired - Fee Related CA2122203C (en) 1993-05-11 1994-04-26 Decontamination of nucleic acid amplification reactions

Country Status (5)

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US (1) US5536649A (en)
EP (1) EP0624643A3 (en)
JP (1) JP2527533B2 (en)
CA (1) CA2122203C (en)
SG (1) SG44809A1 (en)

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Also Published As

Publication number Publication date
US5536649A (en) 1996-07-16
JP2527533B2 (en) 1996-08-28
CA2122203A1 (en) 1994-11-12
SG44809A1 (en) 1997-12-19
EP0624643A2 (en) 1994-11-17
EP0624643A3 (en) 1995-02-22
JPH06319599A (en) 1994-11-22

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