Determination of SNP allelic frequencies using temperature gradient electrophoresis

• US 20050064473A1
• Filed: 08/02/2004
• Published: 03/24/2005
• Est. Priority Date: 07/31/2003
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Wu, Yiqiong, Liu, Zhaowei
• Granted Patent: US 7,303,879 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2565/131 Single/double strand conformational analysis, i.e. SSCP/DSCP

Abstract

A method for determining a frequency of single nucleotide polymorphism (SNP) within genomic DNA includes providing genomic DNA of each of a plurality of different organisms. The genomic DNA of each organism includes first and second portions, e.g., first and second strands. First and second amplicons are prepared from the genomic DNA of each organism. The first amplicon corresponds to the first portion of the genomic DNA and the second amplicon corresponds to the second portion of the genomic DNA. A plurality of duplexes is prepared from the first and second amplicons of the genomic DNA of each organism. At least some of the duplexes include a portion of one of the first amplicons and a portion of one of the second amplicons. The duplexes are subjected to temperature gradient electrophoresis to obtain first electrophoresis data indicative of the rate of SNP at a first location in the genomic DNA of the plurality of organisms.

Claims

1. A method for determining a frequency of single nucleotide polymorphism (SNP) at a first location within genomic DNA, comprising:

providing genomic DNA of each of a plurality of different organisms, the genomic DNA of each organism comprising first and second portions;

preparing, from the genomic DNA of each organism, first and second amplicons, for each organism (i) the first amplicon corresponding to the first portion of the genomic DNA and (ii) the second amplicon corresponding to the second portion of the genomic DNA;

preparing, from the first and second amplicons of the genomic DNA of each organism, a plurality of duplexes, at least some of the duplexes comprising (i) a portion of one of the first amplicons and (ii) a portion of one of the second amplicons; and

subjecting the duplexes to temperature gradient electrophoresis to obtain first electrophoresis data indicative of the rate of SNP at the first location in the genomic DNA of the plurality of organisms.

2. The method of claim 1, wherein, for each organism, the first portion of the genomic DNA corresponds to a first copy of a first portion of a gene of the organism and the second portion of the genomic DNA corresponds to a second copy of the first portion of the gene of the organism.

3. The method of claim 2, wherein, for each organism, the first amplicon comprises at least one portion corresponding to the first portion of the genomic DNA and the second amplicon comprises at least one portion corresponding to the second portion of the genomic DNA.

4. The method of claim 3, wherein, for each organism, the first amplicon comprises first and second complementary polynucleotide strands, the first and second polynucleotide strands corresponding to complementary portions of the first portion of the genomic DNA.

5. The method of claim 3, wherein, for each organism, the second amplicon comprises first and second complementary polynucleotide strands, the first and second polynucleotide strands corresponding complementary portions of the second portion of the genomic DNA.

6. The method of claim 2, wherein the organisms are mammals.

7. The method of claim 6, wherein the organisms are humans.

8. The method of claim 2, wherein the organisms are plants.

9. The method of claim 2, wherein the organism are microbes.

10. The method of claim 1, wherein, for at least some of the organisms, preparing the plurality of duplexes comprises:

providing the first and second amplicons in denatured form; and

concomitantly annealing the denatured first and second amplicons of each organism.

11. The method of claim 10, comprising, prior to the step of concomitantly annealing, preparing a mixture comprising first and second amplicons of the genomic DNA of each of at least 2 of the organisms.

12. The method of claim 1, comprising, prior to the step of preparing, from the genomic DNA of each organism, first and second amplicons, preparing a mixture comprising genomic DNA of at least 2 of the organisms.

13. The method of claim 1, further comprising determining the rate of SNP in the genomic DNA of the plurality of organisms, the step of determining comprising using the first electrophoresis data.

14. The method of claim 1, wherein the step of prior to the step of preparing, from the genomic DNA of each organism, first and second amplicons, comprises a polymerase chain reaction.

15. The method of claim 14, wherein the polymerase chain reaction is a multiplex reaction.

16. The method of claim 13, wherein the step of determining comprises comparing the first electrophoresis data with second electrophoresis data obtained from temperature electrophoresis of a control sample comprising control polynucleotides having at least a first genotype.

17. The method of claim 16, wherein the first genotype is known.

18. The method of claim 17, wherein at least some of the control polynucleotides are heterozygous.

19. The method of claim 13, wherein the step of determining comprises determining a value indicative of the number of duplexes comprising a mismatch and a second value indicative of a number of duplexes not comprising a mismatch.

20. The method of claim 19, wherein determining the value indicative of the number of duplexes comprising a mismatch comprises determining at least one of:

a number of one or more peaks present within a portion of the first electrophoresis data, an area of one or more peaks present within a portion of the first electrophoresis data, and an intensity of one or more peaks present within a portion of the first electrophoresis data.

21. The method of claim 1, comprising preparing calibration data, the calibration data prepared from temperature gradient electrophoresis of a plurality of control polynucleotides.

22. The method of claim 1, comprising preparing at least a first mixture comprising the first and second amplicons of the genomic DNA of each of at least some of the plurality of organisms and wherein the step of subjecting comprises subjecting the mixture to TGE.

23. The method of claim 22, wherein the step of subjecting the duplexes to temperature gradient electrophoresis comprises an injection step wherein the first and second amplicons are introduced to a separation lane and an electrophoresis step and further wherein the step of preparing the mixture is performed prior to the step of injection.

24. The method of claim 1, wherein the different organisms are different members of the same species of organism.

25. The method of claim 1, comprising forming a mixture comprising genomic DNA of the plurality of organisms.