METHOD FOR IDENTIFYING A NUCLEIC ACID IN A SAMPLE

• US 20110171651A1
• Filed: 01/13/2010
• Published: 07/14/2011
• Est. Priority Date: 01/13/2010
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Richmond, Gregory
• Granted Patent: US 9,758,817 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2563/167 Mass label
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer

Abstract

A method of sample analysis is provided. In certain embodiments, the method may comprise: contacting a nucleic acid sample with a first primer and a second primer under PCR conditions to produce a double stranded product, wherein the second primer comprises a first label and is 5′ blocked; b) contacting the double stranded product with an exonuclease to degrade one strand of the double-stranded product to produce a single stranded product; c) contacting the single stranded product with a third primer under primer extension conditions, wherein the third primer comprises a second label; and d) detecting the first and second labels of the partial duplex. A kit for practicing the method is also provided.

Claims

1. A method of sample analysis comprising:

a) contacting a nucleic acid sample with a first primer and a second primer under PCR conditions to produce a double stranded product, wherein said second primer comprises a first label and is 5′

blocked;

b) contacting said double stranded product with an exonuclease to degrade one strand of said double-stranded product to produce a single-stranded product, wherein said the strand that is degraded is an extension product of the first primer;

c) contacting the single-stranded product with a third primer under primer extension conditions, wherein said third primer comprises a second label and wherein said contacting results in hybridization of the third primer to the single stranded product and extension thereof using said single stranded product as a template to produce a partial duplex; and

d) detecting the first and second labels of said partial duplex.

2. The method of claim 1, wherein said method comprises, between steps c) and d), separating said partial duplex from any single stranded nucleic acid.

3. The method of claim 1, wherein said second primer is 5′

blocked by said first label.

4. The method of claim 1, wherein said partial duplex comprises:

a) a single stranded segment region downstream of the binding site for the second primer; and

b) a double stranded segment comprising;

i. the extension product of said third primer and ii. a region upstream of the binding site for the second primer in said single stranded product.

5. The method of claim 1, wherein said double stranded segment is of at least 50 base pairs in length.

6. The method of claim 1, wherein said first and second labels are distinguishable fluorophores.

7. The method of claim 1, wherein said first and second labels are distinguishable mass tags.

8. The method of claim 7, wherein said method further comprises comprising cleaving said mass tags from said partial duplex to produce cleaved mass tags, prior to detection.

9. The method of claim 8, wherein said mass tags are cleaved from said partial duplex by ultra-violet light.

10. The method of claim 8, wherein said detecting comprises detecting said cleaved mass tags by mass spectrometry.

11. The method of claim 1, wherein steps a), b) and c) are performed sequentially in a single tube.

12. The method of claim 1, wherein said method is a multiplex assay that employs:

multiple pairs of first and second primers, each second primer comprising a distinguishable first label; and

multiple third primers comprising second labels that are distinguishable from one another and from said first labels.

13. The method of claim 12, wherein said multiplex assay produces multiple different partial duplexes that are separated other nucleic acid by chromatography prior to cleavage and subsequent detection of said first and second labels.

14. The method of claim 1, wherein said exonuclease is X, exonuclease.

15. The method of claim 1, wherein no snap-cooling is performed between steps a) and c) of said method.

16. The method of claim 1, wherein detection of the first and second labels indicates that said sample comprises a nucleic acid from a particular species.

17. A kit comprising:

a) a first primer;

b) a second primer that comprises a first label and that is 5′

end blocked;

c) a third primer that comprises a second label;

d) reagents for performing a PCR reaction using said first and second primers; and

e) an exonuclease that degrades PCR-generated extension products of said first primer;

wherein said first and second primers, when employed in a PCR reaction using a first template, produce a double stranded product, one strand of which is degradable using said exonuclease to produce a single stranded product; and

wherein said third primer, when employed in a primer extension reaction, hybridizes to said single stranded product and is extended using said single stranded product as a template.

18. The kit of claim 17, wherein said first and second primers are mixed with said reagents in said kit.

19. The kit of claim 17, wherein said reagents are in a different vessel relative to said first and second primers.

20. The kit of claim 17, wherein said first and second labels are mass tags.