Allele-specific sequence variation analysis

• US 20050089904A1
• Filed: 09/02/2004
• Published: 04/28/2005
• Est. Priority Date: 09/05/2003
• Status: Active Grant

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Inventors: Beaulieu, Martin, Van Den Boom, Dirk J.
• Granted Patent: US 9,394,565 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 2565/627 being a mass spectrometer

Abstract

Fragmentation-based methods and systems, particularly mass spectrometry based methods and systems, for the analysis of sequence variations including haplotypes are provided. Also provided are methods for obtaining a specific allele from a nucleic acid mixture, such as genomic DNA.

Claims

1. A method for sequence variation analysis, comprising:

a) fragmenting a target nucleic acid, or a nucleic acid product transcribed from the target nucleic acid at a plurality of specific and predictable sites;

b) fragmenting or simulating fragmentation of a reference target nucleic acid into fragments at the same plurality of specific and predictable sites of step a); and

c) genotyping the nucleic acid sequence corresponding to a single or specific allele at one or more loci.

2. The method of claim 1, further comprising a step of, prior to fragmenting the target nucleic acid, modifying a mixture of nucleic acids to obtain a target nucleic acid sequence corresponding to a single or specific allele.

3. The method of claim 1, wherein the sequence variation analysis is selected from genotyping, haplotyping, HLA-typing or re-sequencing.

4. The method of claim 1, further comprising a step of amplifying the target nucleic acid to obtain a specific allele.

5. The method of claim 4, wherein the amplification step is conducted by Allele-Specific PCR amplification.

6. The method of claim 4, wherein the amplification step is PCR amplification from a diluted DNA sample containing an amount of haploid genome equivalents selected from 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1.

7. The method of claim 4, wherein the amplification step is conducted in the presence of a competitor oligonucleotide that is non-extendable.

8. The method of claim 4, wherein the amplification step is conducted in the presence of a competitor oligonucleotide that is exonuclease resistant.

9. The method of claim 7, wherein the competitor oligonucleotide comprises a modified 3′

-end nucleotide.

10. The method of claim 9, wherein the modified 3′

-end nucleotide is a modification selected from an inverted base, a dideoxynucleotide, a ribonucleotide, a 3′

Phosphate, a carbon chain spacer, or a peptide nucleic acid (PNA).

11. The method of claim 9, wherein the modified 3′

-end nucleotide of the competitor oligonucleotide is bound to the previous nucleotide via a phosphorothioate or methylphosphonate link.

12. The method of claim 7, wherein the competitor oligonucleotide is added to the amplification reaction in an excess amount over a concentration of an AS-PCR amplification primer selected from among:

1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold or 75-fold or more.

13. The method of claim 1, wherein the target nucleic acid sequence corresponding to a single or specific allele has a specificity of an amount selected from greater than 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% of the desired allele relative to the undesired allele.

14. The method of claim 4, wherein the amplification step is conducted in the presence of at least one oligonucleotide primer that comprises a promoter sequence.

15. The method of claim 4, wherein the amplification step includes amplification with an RNA polymerase.

16. The method of claim 5, wherein the amplification step is performed in the presence of a thermostable DNA polymerase, or a mixture of thermostable DNA polymerases, having exonuclease activity.

17. The method of claim 1, wherein the specific and predictable fragmentation is base-specific fragmentation.

18. The method of claim 17, wherein fragmenting steps a) and/or b) are conducted by contacting the target nucleic acid with one or more specific cleavage reagents.

19. The method of claim 1, wherein the genotyping step comprises:

d) determining mass signals of the fragments produced in a) and b);

e) determining differences in the mass signals between the fragments produced in a) and the fragments produced in b); and

f) determining a reduced set of sequence variation candidates from the differences in the mass signals and thereby determining sequence variations in the target nucleic acid compared to the reference nucleic acid.

20. The method of claim 1, wherein the target nucleic acid is selected from the group consisting of DNA, cDNA, and RNA.

21. The method of claim 19, further comprising scoring the candidate sequences and determining the sequence variations in the target nucleic acid molecule.

22. The method of claim 21, wherein determining sequence variations comprises:

g) identifying fragments that are different between the target nucleic acid and the reference nucleic acid;

h) determining compomers corresponding to the identified different fragments in step h) that are compomer witnesses; and

c) determining a reduced set of sequence variations corresponding to the compomer witnesses that are candidate sequences to determine the sequence variations in the target nucleic acid compared to the reference nucleic acid.

23. The method of claim 19, wherein the differences in output signals are manifested as missing signals, additional signals, signals that are different in intensity, and/or as having a different signal-to-noise ratio.

24. The method of claim 19, wherein the masses are determined by mass spectrometry.

25. The method of claims 19, wherein the sequence variation is selected from the group consisting of a mutation, a polymorphism, an insertion, a deletion, a substitution, and a single nucleotide polymorphism.

26. The method of claim 1, wherein the target nucleic acid molecule is from an organism selected from the group consisting of eukaryotes, prokaryotes and viruses.

27. The method of claim 26, wherein the organism is a human.

28. The method of claim 18, wherein a specific cleavage reagent is an RNAse.

29. The method of claim 28, wherein the specific cleavage reagent is selected from RNase T₁, RNase U₂, the RNase PhyM, RNase A, chicken liver RNase (RNase CL3) and cusavitin.

30. The method of claim 18, wherein a specific cleavage reagent is a DNA glycosylase-mediated.

31. The method of claim 19, wherein sequence variations in the target nucleic acid permit genotyping or haplotyping a subject, forensic analysis, disease diagnosis or disease prognosis.

32. A method for determining a nucleic acid genotype or haplotype, comprising:

amplifying a target nucleic acid in the presence of a competitor oligonucleotide that is non-extendable and is exonuclease resistant; and

genotyping the target nucleic acid.

33. The method of claim 32, wherein the competitor oligonucleotide is non-extendable by a polymerase.

34. The method of claim 32, wherein the competitor oligonucleotide comprises a modified 3′

-end nucleotide.

35. The method of claim 34, wherein the modified 3′

-end nucleotide is a modification selected from an inverted base, a dideoxynucleotide, a ribonucleotide, a 3′

Phosphate, a carbon-chain spacer, or a PNA.

36. The method of claim 34, wherein the modified 3′

-end nucleotide of the oligonucleotide is bound to the previous nucleotide via a phosphorothioate or methylphosphonate link.

37. The method of claim 32, wherein the competitor oligonucleotide is added to the amplification reaction in an excess amount over a concentration of an AS-PCR amplification primer selected from among:

1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold or 75-fold or more.

38. The method of claim 32, wherein the amplification step is conducted in the presence of at least one oligonucleotide primer that comprises a promoter sequence.

39. The method of claim 33, wherein the polymerase is an RNA polymerase.

40. The method of claim 32, wherein the amplification is performed in the presence of a thermostable DNA polymerase, or a mixture of thermostable DNA polymerases, having exonuclease activity.

41. The method of claim 32, wherein the genotyping step is conducted using base-specific fragmentation.

42. The method of claim 32, wherein the genotyping step further comprises mass spectrometric analysis nb.