Mass spectrometric methods for detecting mutations in a target nucleic acid
View Patent ↗Fast and highly accurate mass spectrometry-based processes for detecting particular nucleic acid molecules and mutations in the molecules are provided. In some embodiments, a process comprises: amplifying one or more target nucleic acid molecules from a biological sample; ionizing and volatilizing the amplified product; and analyzing a product by mass spectrometry to determine an observed molecular mass of the product.
1. A process for characterizing a target nucleic acid from a biological sample, comprising:
a) amplifying a target nucleic acid from a biological sample by polymerase chain reaction (PCR);
b) ionizing and volatilizing the product of step (a); and
c) analyzing (i) a forward strand PCR product by mass spectrometry to determine an observed molecular mass of the forward strand PCR product, or (ii) analyzing a reverse strand PCR product by mass spectrometry to determine an observed molecular mass of the reverse strand PCR product;
whereby the target nucleic acid is characterized based on the analysis of (c).
2. The process of claim 1 , wherein step (c) comprises analyzing (i) a forward strand PCR product by mass spectrometry to determine an observed molecular mass of the forward strand PCR product, and (ii) analyzing a reverse strand PCR product by mass spectrometry to determine an observed molecular mass of the reverse strand PCR product.
3. The process of claim 1 , wherein the target nucleic acid comprises at least one mutation.
4. The process of claim 1 , wherein the biological sample comprises material obtained from a human, animal, plant, fungus, parasite or virus.
5. The process of claim 4 , wherein characterizing the target nucleic acid identifies different individuals or strains within the same species.
6. The process of claim 1 , wherein the biological sample comprises material obtained from a human.
7. The process of claim 1 , wherein the biological sample comprises material obtained from an infectious organism.
8. The process of claim 7 , wherein the target nucleic acid is a sequence present in an infectious organism and distinct from host cell sequences.
9. The process of claim 1 , wherein the product of step (a) is conditioned.
10. The process of claim 9 , wherein said conditioning comprises cation exchange.
11. The process of claim 10 , wherein the target nucleic acid is subjected to cation exchange directly after step (a), and step (b) is performed directly after the cation exchange.
12. The process of claim 1 , wherein steps b) and c) are performed directly after step a).
13. The process of claim 1 , wherein step c) comprises the use of electrospray (ES) mass spectrometry.
14. The process of claim 1 , wherein step c) comprises the use of matrix assisted laser desorption ionization (MALDI) mass spectrometry.
15. The process of claim 1 , wherein the target nucleic acid is not specifically digested.
16. The process of claim 15 , wherein the target nucleic acid is not specifically digested by a nuclease.
17. The process of claim 1 , wherein the target nucleic acid is double stranded.
18. The process of claim 1 , wherein the target nucleic acid is DNA.
19. The process of claim 1 , wherein, in solution, (i) the target nucleic acid is isolated from the biological sample, and (ii) the isolated target nucleic acid directly is subjected to step (a).
20. The process of claim 1 , wherein the biological sample comprises cells, and the target nucleic acid is isolated from the biological sample by lysing the cells.
21. The process of claim 1 , which comprises collecting the biological sample.
22. The process of claim 1 , which comprises isolating the target nucleic acid from the biological sample.
23. A process for characterizing a target nucleic acid from a biological sample, comprising:
a) providing a biological sample;
b) isolating a target nucleic acid from the biological sample;
c) amplifying the target nucleic acid of (b) by polymerase chain reaction (PCR);
d) ionizing and volatilizing the product of step (c); and
e) analyzing (i) a forward strand PCR product by mass spectrometry to determine an observed molecular mass of the forward strand PCR product, or (ii) analyzing a reverse strand PCR product by mass spectrometry to determine an observed molecular mass of the reverse strand PCR product;
whereby the target nucleic acid is characterized based on the analysis of (e).
24. The process of claim 23 , wherein step (e) comprises analyzing (i) a forward strand PCR product by mass spectrometry to determine an observed molecular mass of the forward strand PCR product, and (ii) analyzing a reverse strand PCR product by mass spectrometry to determine an observed molecular mass of the reverse strand PCR product.
25. The process of claim 23 , wherein two or more biological samples are provided and the nucleic acid is isolated from the two or more biological samples.
26. The process of claim 23 , wherein the target nucleic acid comprises at least one mutation.
27. The process of claim 23 , wherein the biological sample comprises material obtained from a human, animal, plant, fungus, parasite or virus.
28. The process of claim 27 , wherein characterizing the target nucleic acid identifies different individuals or strains within the same species.
29. The process of claim 23 , wherein the biological sample comprises material obtained from a human.
30. The process of claim 23 , wherein the biological sample comprises material obtained from an infectious organism.
31. The process of claim 29 , wherein the target nucleic acid is a sequence present in an infectious organism and distinct from host cell sequences.
32. The process of claim 23 , wherein the product of step (c) is conditioned.
33. The process of claim 32 , wherein said conditioning comprises cation exchange.
34. The process of claim 33 , wherein the target nucleic acid is subjected to cation exchange directly after step (c), and step (d) is performed directly after the cation exchange.
35. The process of claim 23 , wherein steps d) and e) are performed directly after step c).
36. The process of claim 23 , wherein step e) comprises the use of electrospray (ES) mass spectrometry.
37. The process of claim 23 , wherein step e) comprises the use of matrix assisted laser desorption ionization (MALDI) mass spectrometry.
38. The process of claim 23 , wherein the target nucleic acid is not specifically digested.
39. The process of claim 38 , wherein the target nucleic acid is not specifically digested by a nuclease.
40. The process of claim 23 , wherein the target nucleic acid is double stranded.
41. The process of claim 23 , wherein the target nucleic acid is DNA.
42. The process of claim 23 , wherein step (c) is performed directly after step (b) and each step is performed in solution.
43. The process of claim 23 , wherein the biological sample comprises cells, and the target nucleic acid molecule is isolated from the biological sample by lysing the cells.
44. The process of claim 1 , wherein the product of step (a) is from about 7 to about 50 bases.
45. The process of claim 1 , wherein the product of step (a) is about 40 bases, about 50 bases, about 100 bases, or about 200 bases.
46. The process of claim 23 , wherein the product of step (a) is from about 7 to about 50 bases.
47. The process of claim 23 , wherein the product of step (a) is about 40 bases, about 50 bases, about 100 bases, or about 200 bases.