Sample generation for genotyping by mass spectrometry

• US 20040038234A1
• Filed: 05/01/2003
• Published: 02/26/2004
• Est. Priority Date: 06/30/2000
• Status: Abandoned Application

• Current Assignee: Inserm (institut national de la sant et de la recherche mdicale
• Original Assignee: Centre National De Genotypage, Inserm (institut national de la sant et de la recherche mdicale
• Inventors: Lechner, Doris, Sauer, Sascha, Gut, Ivo Glynne
• Application Number:
  US10/311,331
• Publication Number:
  US 20040038234A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The invention is drawn to a universal method for preparing DNA samples for genotyping of single nucleotide polymorphisms (SNP), deletions and insertions by mass spectrometry. The invention relates to a method for genotyping that comprises three steps: a) reducing the complexity of the genome, b) generating allele specific products, c) analyzing the products by mass spectrometry, without purifying the products obtained in step b).

Claims

1. A method for DNA genotyping by mass spectrometry, comprising the steps of:

a. reduction of the complexity of the DNA sample,

b. generation of allele specific products on the products generated in step a, wherein the generation of allele specific products in step b. is achieved by at least one method that uses (an) allele specific oligonucleotide(s)

c. mass spectrometric analysis of the products generated in step b, wherein the mass spectrometric analysis in step c. is performed on the products generated in step b. without purification or separation from the reaction mixture.

2. The method of claim 1, wherein step b. is performed by at least one method chosen in the group consisting of primer extension, allele specific ligation, and cleavage reaction.

3. The method of claim 1 or 2, wherein step a is performed by at least one method chosen in the group consisting of Alu PCR, DOP PCR, LCR, AFLP, generation of padlock probes, and cleavage reaction.

4. The method of any of claims 1 to 3, wherein the allele specific oligonucleotide(s) is (are) modified as to allow a mass spectrometric analysis with significantly higher sensitivity than for (an) unmodified oligonucleotide(s).

5. The method of any of claims 1 to 4, wherein the products generated in step b. are or can be conditioned to carry a single excess charge, either positive or negative.

6. The method of any of any of claims 1 to 5, wherein the products generated in step b. and after conditioning are between 2 and 10 bases long.

7. The method of any of claims 1 to 6, wherein at least one oligonucleotide used in step b. is chimeric.

8. The method of claim 7, wherein the chimeric oligonucleotide(s) consists in a regular sugar-phosphate backbone with an end being a phosphorothioate, phosphoroselenoate, methylphosphonate, ethylphosphonate, methoxyphosphonate, ethoxyphosphonate, peptide nucleic acid or a peptide.

9. The method of claim 8, wherein the regular part of the oligonucleotide(s) is between 13 and 40 bases long.

10. The method of any of claims 7 to 9, wherein the products generated in step b. are obtained by cleavage of the modified part from the unmodified part of the chimeric oligonucleotide(s).

11. The method of claim 10, wherein the cleavage is performed by at least one method of the group consisting of an inhibited endonuclease digest, an inhibited exonuclease digest or chemical cleavage.

12. The method of any of claims 1 to 11, wherein step c. is performed by mass analysis of the products of step b., the mass of each generated product being unique and allowing unambiguous allele assignment.

13. The method of any of claims 1 to 12, wherein step c. is performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

14. The method of claim 13, wherein the matrix is chosen from the group consisting of α

-cyano-4-hydroxycinnamic acid, α

-cyano-4-hydroxycinnamic acid methyl ester, α

-cyano4-methoxycinnamic acid matrices, derivatives thereof, and a mixture of these matrices.

15. The method of any of claims 1 to 12, wherein step c. is performed by electrospray ionization mass spectrometry.