Methods for rapid identification and quantitation of nucleic acid variants

• US 20070218467A1
• Filed: 07/21/2006
• Published: 09/20/2007
• Est. Priority Date: 07/21/2005
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Hall, Thomas, Hofstadler, Steven, Ecker, David, Lowery, Kristin
• Granted Patent: US 8,026,084 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 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2531/113 PCR
• 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 2545/10 the purpose being quantitative analysis
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

There is a need for nucleic acid analysis which is both specific and rapid, and in which no nucleic acid sequencing is required. The present invention addresses this need, among others by providing a method of nucleic acid amplification of overlapping sub-segments of a nucleic acid followed by molecular mass measurement of resulting amplification products by mass spectrometry, and determination of the base compositions of the amplification products.

Claims

1. A method for analyzing a nucleic acid comprising the steps of:

(a) obtaining a sample comprising nucleic acid for base composition analysis;

(b) selecting at least two primer pairs that will generate overlapping amplification products of at least two sub-segments of the nucleic acid;

(c) amplifying at least two nucleic acid sequences of a region of the nucleic acid designated as a target for base composition analysis using the at least two primer pairs, thereby generating at least two overlapping amplification products;

(d) determining base compositions of the amplification products by;

(i) measuring molecular masses of one or more of the amplification products generated in step (c) using a mass spectrometer; and

(ii) converting one or more of the measured molecular masses to base compositions;

(e) comparing one or more of the base compositions with a source of reference base composition data for the nucleic acid sequence; and

(f) identifying the presence of a particular nucleic acid sequence or variant thereof.

2. The method of claim 1 wherein the step of selecting at least two primer pairs is selecting at least one triplex combination of primer pairs.

3. The method of claim 1 wherein the amplification product is from about 40 nucleobases in length to about 150 nucleobases in length.

4. The method of claim 3 wherein the amplification product is from about 46 nucleobases in length to about 140 nucleobases in length.

5. The method of claim 1 wherein the amplification product is less than 130 nucleobases.

6. The method of claim 1 wherein the amplification product is from about 85 nucleobases in length to about 140 nucleobases in length.

7. The method of claim 1 wherein the sample comprising nucleic acid for base composition analysis is selected from the group consisting of a human chromosomal nucleic acid, a human mitochondrial nucleic acid, a bacterial nucleic acid, a viral nucleic acid, a fungal nucleic acid, a synthetic nucleic acid, a recombinant nucleic acid and a combination thereof.

8. The method of claim 1 wherein the confirming step identifies at least one variant whose base composition differs from the base composition of its corresponding reference sub-segment, thereby characterizing a genotype of a human, bacterium, virus or fungus.

9. The method of claim 1 wherein the identifying step identifies at least one amplification product whose base composition differs from the base composition of its corresponding reference sub-segment, thereby identifying a genetically-engineered bacterium virus or fungus.

10. The method of claim 7 wherein the sample for base composition analysis is at least a portion of the HV1 segment of a mitochondrial DNA.

11. The method of claim 10 wherein the HV1 segment is nucleotide positions 15924 to 16451 of SEQ ID NO:

51.

12. The method of claim 10 wherein the sample for base composition analysis comprises a heteroplasmy variant.

13. The method of claim 7 wherein the sample for base composition analysis is at least a portion of the HV2 segment of a mitochondrial DNA.

14. The method of claim 13 wherein the HV2 segment is nucleotide positions 31 to 576 of SEQ ID NO:

51

15. The method of claim 13 wherein the sample for base composition comprises a heteroplasmy variant.

16. The method of claim 1 wherein the amplifying step is carried out in the presence of a dNTP comprising a mass modifying tag.

17. The method of claim 16 wherein the dNTP is 2′

-deoxy-guanosine-5′

-triphosphate.

18. The method of claim 16 wherein the dNTP is 13.sup.C.

19. The method of claim 7 wherein the sample for base composition analysis is mitochondrial DNA and the at least two primers comprise combinations of SEQ ID NOs:

1;

26, 2;

27, 3;

28, 4;

29, 5;

30, 6;

31, 7;

32, 8;

33, 9;

34, 10;

35, 11;

36, 12;

37, 13;

38, 14;

39, 15;

40, 16;

41, 17;

42, 18;

43, 19;

44, 20;

45, 21;

46, 22;

47, 23;

48, 24;

49, and 25;

50.

20. The method of claim 7 wherein the sample for base composition analysis is human mitochondrial DNA and the obtained base compositions are compared with corresponding reference sub-segments of a reference sequence comprising the Anderson/Cambridge sequence (SEQ ID NO:

51).

21. The method of claim 7 wherein the comparing of the measured base composition to the reference sub-segments provides genotype information comprising human identification, SNP identification, VNTR identification, recombinant insert identification, heteroplasmy identification, genetic disease disposition and combinations thereof.

22. The method of claim 1 wherein the amplification step is quantitative and further comprises the step of adding to the sample a known quantity of a calibrant that will co-amplify with the sample for sequence identity analysis.

23. The method of claim 1 wherein the mass spectrometry is electrospray Fourier transform ion cyclotron resonance mass spectrometry or electrospray time-of-flight mass spectrometry.

24. The method of claim 1 wherein the target region for base composition analysis has a length falling in the range comprising an upper limit of about 700 nucleobases and comprising a lower limit of about 300 nucleobases.

25. The method of claim 24 wherein the range comprises an upper limit of about 400 nucleobases and comprises a lower limit of about 600 nucleobases.

26. The method of claim 2 wherein the sample for base composition analysis is mitochondrial DNA and the triplex combination of primer pairs is selected from the group consisting of primer pairs 4:

29, 12;

37 and 17;

42;

primer pairs 3;

28, 19;

44 and 25;

50;

primer pairs 2;

27, 11;

36 and 18;

43;

primer pairs 10;

35, 1;

26 and 24;

49;

primer pairs 13;

38, 20;

45 and 5;

30;

primer pairs 13;

38,20;

45 and 6;

31;

primer pairs 23;

48, 9;

34 and 5;

30;

primer pairs 15;

40, 8;

33 and 22;

47; and

primer pairs 7;

32, 21;

46 and 16;

41.

27. The method of claim 1 wherein the nucleic acid is a vector.

28. The method of claim 27 wherein the vector comprises a gene encoding a therapeutic protein.

29. A method for identifying a human comprising the steps of:

(a) obtaining a sample comprising mitochondrial DNA of the human for base composition analysis;

(b) selecting at least two primer pairs that will generate overlapping amplification products representing sub-segments of the mitochondrial DNA;

(c) amplifying at least two nucleic acid sequences of a region of the mitochondrial DNA designated as a target for base composition analysis using the at least two primer pairs, thereby generating at least two overlapping amplification products;

(d) determining base compositions of the amplification products by;

(i) measuring molecular masses of one or more of the amplification products generated in step (c) using a mass spectrometer; and

(ii) converting one or more of the measured molecular masses to base compositions;

(e) comparing one or more of the base compositions with a source of reference base composition data for the nucleic acid sequence thereby identifying the human.

30. The method of claim 29 wherein the sample for base composition analysis is at least a portion of the HV1 segment of a mitochondrial DNA.

31. The method of claim 30 wherein the HV1 segment is nucleotide positions 15924 to 16451 of SEQ ID NO:

51.

32. The method of claim 29 wherein the sample for base composition analysis is at least a portion of the HV2 segment of a mitochondrial DNA.

33. The method of claim 32 wherein the HV2 segment is nucleotide positions 31 to 576 of SEQ ID NO:

51

34. The method of claim 29 wherein the source of reference base composition is a head-to-head comparison.

35. The method of claim 29 wherein the source of reference base composition is a standard reference database.

36. A method for characterizing heteroplasmy in mitochondrial DNA comprising the steps of:

(a) obtaining a sample comprising mitochondrial DNA for base composition analysis;

(b) selecting at least two primer pairs that will generate overlapping amplification products representing sub-segments of the mitochondrial DNA;

(c) amplifying at least two nucleic acid sequences of a region of the mitochondrial DNA designated as a target for base composition analysis using the at least two primer pairs, thereby generating at least two overlapping amplification products;

(d) determining base compositions of the amplification products by;

(i) measuring molecular masses of one or more of the amplification products generated in step (c) using a mass spectrometer; and

(ii) converting one or more of the measured molecular masses to base compositions;

(e) comparing one or more of the base compositions with one or more base compositions of reference sub-segments of a reference sequence; and

(f) identifying at least two distinct amplification products with distinct base compositions obtained by the same pair of primers, thereby characterizing the heteroplasmy.