Method for genotyping of single nucleotide polymorphism

• US 6,479,242 B1
• Filed: 10/27/2000
• Issued: 11/12/2002
• Est. Priority Date: 10/26/2000
• Status: Expired due to Fees

• Current Assignee: Cleveland State University
• Original Assignee: Cleveland State University
• Inventors: Sun, Xiyuan, Guo, Baochuan
• Primary Examiner(s): Campbell, Eggerton A.
• Assistant Examiner(s): Chunduru, Suryaprabha
• Application Number:
  US09/698,505
• Time in Patent Office: 746 Days
• Field of Search: 435/6, 435/91.2, 536/22.1
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The VSET method comprises: providing a polynucleotide acid sample comprising at least one target site, and a first region of nucleotides immediately adjacent to the target site; preferably genomic DNA; preferably amplifying the polynucleotide; then combining the polynucleotide sample with: three dideoxynucletides selected from the group of ddGTP, ddATP, ddCTP, and ddTTP; and one deoxynucleotide selected from the group consisting of and dGTP, dATP, dCTP, and dTTP wherein the nucleotide of the deoxynucleotide is not the same as the nucleotide in the dideoxynulceotide; and a mini-sequencing primer complementary to the first region of nucleotides; extending the mini-sequencing primer with a dideoxynucletide or deoxynulceotide whose base is complementary to the base at the target site, to provide extension products; and then identifying the extension products, preferably by (MALDI-TOF) mass spectrometry. One of the three ddNTps is complementary to one of the allelic variations at the single point mutation site while the deoxynucleotide is complementary to the other allelic variant at the single point mutation site. The mini-sequencing primer hybridizes to a polynucleotide sequence immediately next to a single point mutation site and is extended by the DNA polymerases. The genotype at the variable site is determined on the basis of the number of nucleotides contained in the extension products.

Claims

1. A method for determining a nucleotide in a polynucleotide, comprising the following steps:

a. providing a polynucleotide comprising at least one target site, and a first region of nucleotides immediately adjacent to the target site;

b. then combining the polynucleotide with;

a minisequencing primer complementary to the first region of nucleotides;

three dideoxynucletides selected from the group consisting of ddATP, ddCTP, ddTTP and ddGTP;

a deoxynucleotide selected from the group consisting of dATP, dCTP, dTTP and dGTP;

wherein the nucleotide of the deoxynucleotide is not the same as the nucleotide in the dideoxynulceotide; and

c. then extending the mini-sequencing primer with dideoxynucletide or deoxynulceotide whose base is complementary to the base at the target site, to provide extension products; and

d. then analyzing the extension products with mass spectrometry.

2. The method of claim 1, wherein the polynucleotide is amplified before step b.

3. The method of claim 1, further comprising the step of desalting after mini-sequencing.

4. The method of claim 1, wherein the nucleotide polymorphism is a single nucleotide polymorphism.

5. The method of claim 4, wherein the single nucleotide polymorphism is 1691^th base of the coagulation Factor V gene.

6. The method of claim 1, wherein the mass spectrometry is matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry.

27. The method of claim 1, wherein the mini-sequencing primers are at least 5 nucleotide bases in length.

7. A method for determining a nucleotide in a single nucleotide polymorphism, comprising the following steps:

a. providing a polynucleotide comprising at least one single nucleotide polymorphism site, and a first region of nucleotides immediately adjacent to the polymorphism site;

b. then combining the polynucleotide with;

a minisequencing primer which is complementary to the first nucleotide region;

three different dideoxynucletides selected from the group consisting of ddATP, ddCTP, ddTTP and ddGTP;

a deoxynucleotide selected from the group consisting of dATP, dCTP, dTTP and dGTP;

wherein the nucleotide of the deoxynucleotide is not the same as the nucleotide in the dideoxynulceotide; and

c. extending the mini-sequencing primer with dideoxynucletide or deoxynulceotide whose base is complementary to the base at the single nucleotide polymorphism site, to provide extension products; and

d. analyzing the extension products with mass spectrometry.

8. The method of claim 7, wherein the polynucleotide is amplified before step b.

9. The method of claim 8, wherein the amplification is by polymerase chain reaction, and the unreacted deoxynucleotides and primers are removed after amplification.

10. The method of claim 7, further comprising the step of desalting after mini-sequencing.

11. The method of claim 10, wherein the desalting is done by ethanol purification.

12. The method of claim 8, wherein the nucleotide polymorphism is a single nucleotide polymorphism.

13. The method of claim 12, wherein the polynucleotide further comprises a second single polymorphism site.

14. The method of claim 7, wherein the mass spectrometry is Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry.

15. The method of claim 7, wherein the polynucleotide is amplified by polymerase chain reaction before step b, the single nucleotide polymorphism is 1691^th base of the coagulation Factor V gene, the mass spectrometry is Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry.

16. A method for determining the nucleotides in multiple nucleotide polymorphism sites, comprising the following steps:

a. providing a polynucleotide sample comprising;

a first polynucleotide comprising at least one single nucleotide polymorphism site, and a first region of nucleotides immediately adjacent to the polymorphism site;

a second polynucleotide comprising at least one single nucleotide polymorphism site, and a second region of nucleotides immediately adjacent to the polymorphism site;

b. then combining the polynucleotide sample with;

a first minisequencing primer which is complementary to the first nucleotide region;

a second minisequencing primer which is complementary to the second nucleotide region;

three different dideoxynucletides selected from the group consisting of ddATP, ddCTP, ddTTP and ddGTP;

a deoxynucleotide selected from the group consisting of dATP, dCTP, dTTP and dGTP;

wherein the nucleotide of the deoxynucleotide is not the same as the nucleotide in the dideoxynulceotide; and

c. extending the mini-sequencing primer with a dideoxynucletide or deoxynulceotide whose base is complementary to the base at the single nucleotide polymorphism site, to provide extension products; and

d. analyzing the extension products with mass spectrometry.

17. The method of claim 16, wherein the polynucleotide sample is amplified before step b.

18. The method of claim 17, wherein the amplification is by polymerase chain reaction.

19. The method of claim 18, wherein unreacted dideoxynucleotides and primers are removed after amplification.

20. The method of claim 17, further comprising the step of desalting after mini-sequencing.

21. The method of claim 20, wherein the desalting is done by ethanol purification.

22. The method of claim 16, wherein the nucleotide polymorphism is a single nucleotide polymorphism.

23. The method of claim 16, wherein the single nucleotide polymorphism is 1691^th base of the coagulation Factor V gene.

24. The method of claim 16, wherein the mass spectrometry is matrix-assisted laser-desorption/ionization time-of-flight the mass spectrometry.

25. The method of claim 17, wherein the polynucleotides are amplified together.

26. The method of claim 17, wherein the polynucleotides are amplified separately, then the amplified polynucleotides are pooled prior to step c.