Mass Spectrometric Methods for Detecting Mutations in a Target Nucleic Acid
Fast and highly accurate mass spectrometry-based processes for detecting particular nucleic acid molecules and mutations in the molecules are provided.
1 - 16 . (canceled)
17 . A process for detecting the presence or absence of a target nucleic acid from a biological sample, comprising:
a) amplifying a nucleic acid from a biological sample;
b) ionizing and volatilizing the product of step a);
c) analyzing the product of step b) by mass spectrometry to determine an observed molecular mass of the product of step b); and
d) comparing the observed molecular mass of the product of step b) to a calculated molecular mass of at least one nucleic acid having a known sequence, wherein the calculated molecular mass of the at least one nucleic acid having a known sequence is derived from the base composition of the at least one nucleic acid having a known sequence;
whereby the presence or absence of the target nucleic acid is detected based on the comparison of (d).
18 . The process of claim 17 , 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.
19 . The process of claim 17 , wherein the target nucleic acid comprises at least one mutation.
20 . The process of claim 17 , wherein the biological sample comprises material obtained from a human, animal, plant, fungus, parasite or virus.
21 . The process of claim 20 , wherein detecting the presence of the target nucleic acid identifies different individuals or strains within the same species.
22 . The process of claim 17 , wherein the biological sample comprises material obtained from a human.
23 . The process of claim 17 , wherein the biological sample comprises material obtained from an infectious organism.
24 . The process of claim 23 , wherein the target nucleic acid is a sequence present in an infectious organism and distinct from host cell sequences.
25 . The process of claim 17 , wherein the product of step (a) is from about 10 to about 200 bases, from about 40 bases to about 200 bases, from about 80 bases to about 200 bases, or from about 100 bases to about 200 bases.
26 . The process of claim 17 , wherein the product of step (a) is about 18 to about 46 bases.
27 . The process of claim 17 , wherein the product of step (a) is conditioned.
28 . The process of claim 27 , wherein said conditioning comprises cation exchange.
29 . The process of claim 28 , wherein the target nucleic acid is subjected to cation exchange directly after step (a), and step (b) is performed directly after the cation exchange.
30 . The process of claim 17 , wherein steps b) and c) are performed directly after step a).
31 . The process of claim 17 , wherein step c) comprises the use of electrospray (ES) mass spectrometry.
32 . The process of claim 17 , wherein step c) comprises the use of matrix assisted laser desorption ionization (MALDI) mass spectrometry.
33 . The process of claim 17 , wherein the target nucleic acid is not specifically digested.
34 . The process of claim 33 , wherein the target nucleic acid is not specifically digested by a nuclease.
35 . The process of claim 17 , wherein the target nucleic acid is double stranded.
36 . The process of claim 17 , wherein the target nucleic acid is DNA.
37 . The process of claim 17 , wherein, in solution, (i) the nucleic acid is isolated from the biological sample, and (ii) the isolated nucleic acid directly is subjected to step (a).
38 . The process of claim 17 , wherein the biological sample comprises cells, and the nucleic acid is isolated from the biological sample by lysing the cells.
39 . The process of claim 17 , which comprises collecting the biological sample.
40 . The process of claim 17 , which comprises isolating the nucleic acid from the biological sample.
41 . The method of claim 17 , wherein RNA is amplified in step a).
42 . The method of claim 17 , wherein the product of step a) is an RNA molecule.
43 . A process for detecting a target nucleic acid from a biological sample, comprising:
a) amplifying a nucleic acid from a biological sample by PCR using at least one pair of primers, wherein said at least one pair of primers comprises a forward primer and a reverse primer;
b) ionizing and volatilizing the product of step a);
c) analyzing a first product of step b) by mass spectrometry to determine an observed molecular mass of the first product of step b), wherein said first product of step b) is the product extended from the forward primer;
d) analyzing a second product of step b) by mass spectrometry to determine an observed molecular mass of the first product of step b), wherein said second product of step b) is the product extended from the reverse primer;
e) comparing the observed molecular mass of the first product of step b) to a calculated molecular mass of a first nucleic acid having a known sequence, wherein the calculated molecular mass of the first nucleic acid having a known sequence is derived from the base composition of the first nucleic acid having a known sequence;
f) comparing the observed molecular mass of the second product of step b) to a calculated molecular mass of a second nucleic acid having a known sequence, wherein the calculated molecular mass of the second nucleic acid having a known sequence is derived from the base composition of the second nucleic acid having a known sequence;
whereby the presence or absence of the target nucleic acid is detected based on the comparisons of (e) and (f).
44 . The process of claim 43 , 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.
45 . The process of claim 43 , wherein the target nucleic acid comprises at least one mutation.
46 . The process of claim 43 , wherein the biological sample comprises material obtained from a human, animal, plant, fungus, parasite or virus.
47 . The process of claim 46 , wherein detecting the presence of the target nucleic acid identifies different individuals or strains within the same species.
48 . The process of claim 43 , wherein the biological sample comprises material obtained from a human.
49 . The process of claim 43 , wherein the biological sample comprises material obtained from an infectious organism.
50 . The process of claim 49 , wherein the target nucleic acid is a sequence present in an infectious organism and distinct from host cell sequences.
51 . The process of claim 43 , wherein the product of step (a) is from about 10 to about 200 bases, from about 40 bases to about 200 bases, from about 80 bases to about 200 bases, or from about 100 bases to about 200 bases.
52 . The process of claim 43 , wherein the product of step (a) is about 18 to about 46 bases.
53 . The process of claim 43 , wherein the product of step (a) is conditioned.
54 . The process of claim 53 , wherein said conditioning comprises cation exchange.
55 . The process of claim 54 , wherein the target nucleic acid is subjected to cation exchange directly after step (a), and step (b) is performed directly after the cation exchange.
56 . The process of claim 43 , wherein steps b) and c) are performed directly after step a).
57 . The process of claim 43 , wherein step c) comprises the use of electrospray (ES) mass spectrometry.
58 . The process of claim 43 , wherein step c) comprises the use of matrix assisted laser desorption ionization (MALDI) mass spectrometry.
59 . The process of claim 43 , wherein the target nucleic acid is not specifically digested.
60 . The process of claim 59 , wherein the target nucleic acid is not specifically digested by a nuclease.
61 . The process of claim 43 , wherein the target nucleic acid is double stranded.
62 . The process of claim 43 , wherein the target nucleic acid is DNA.
63 . The process of claim 43 , wherein, in solution, (i) the nucleic acid is isolated from the biological sample, and (ii) the isolated nucleic acid directly is subjected to step (a).
64 . The process of claim 43 , wherein the biological sample comprises cells, and the nucleic acid is isolated from the biological sample by lysing the cells.
65 . The process of claim 43 , which comprises collecting the biological sample.
66 . The process of claim 43 , which comprises isolating the nucleic acid from the biological sample.