DNA sequencing by mass spectrometry via exonuclease degradation
First Claim
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1. A method of determining a sequence of a nucleic acid, comprising the steps of:
- (i) obtaining the nucleic acid to be sequenced;
(ii) cleaving the nucleic acid to be sequenced from a first end to a second end with an exonuclease to sequentially release individual nucleotides;
(iii) identifying each of the sequentially released nucleotides by mass spectrometry; and
(iv) determining the sequence of the nucleic acid from the identified nucleotides.
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Abstract
The invention provides fast and highly accurate mass spectrometer based processes for directly sequencing a target nucleic acid (or fragments generated from the target nucleic acid), which by means of protection, specificity of enzymatic activity, or immobilization, are unilaterally degraded in a stepwise manner via exonuclease digestion and the nucleotides, derivatives or truncated sequences detected by mass spectrometry.
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Citations
44 Claims
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1. A method of determining a sequence of a nucleic acid, comprising the steps of:
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(i) obtaining the nucleic acid to be sequenced; (ii) cleaving the nucleic acid to be sequenced from a first end to a second end with an exonuclease to sequentially release individual nucleotides; (iii) identifying each of the sequentially released nucleotides by mass spectrometry; and (iv) determining the sequence of the nucleic acid from the identified nucleotides. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A method of determining a sequence of a nucleic acid, comprising the steps of:
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(i) obtaining the nucleic acid to be sequenced; (ii) cleaving the nucleic acid to be sequenced from a first end to a second end with an exonuclease to produce multiple sets of nested nucleic acid fragments; (iii) determining the molecular weight value of each one of the sets of nucleic acid fragments by mass spectrometry; and (iv) determining the sequence of the nucleic acid from the molecular weight values of the sets of nucleic acid fragments. - View Dependent Claims (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40)
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41. A method of nucleic acid sequencing, comprising the steps of:
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(a) obtaining a target nucleic acid to be sequenced wherein the target nucleic acid sequence is flanked by cleavable sites at both ends and wherein the plus and the minus strand of the target nucleic acid are distinguishable from each other by differential mass-modification; (b) cleaving the target nucleic acid at the flanking cleavable sites; (c) denaturing the cleaved target nucleic acid to generate single-stranded plus and minus strands; (d) simultaneously cleaving the denatured plus and minus strands from a first end to a second end with an exonuclease to sequentially release individual nucleotides, wherein the individual nucleotides derived from the plus strand are mass-differentiated from the individual nucleotides derived from the minus strand; (e) identifying each of the sequentially released nucleotides produced in step (d) by mass spectrometry; and (f) determining the sequence of the target nucleic acid from the identified nucleotides. - View Dependent Claims (42)
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43. A method of nucleic acid sequencing, comprising the steps of:
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(a) obtaining a target nucleic acid to be sequenced wherein the target nucleic acid sequence is flanked by cleavable sites at both ends and wherein the plus and the minus strand of the target nucleic acid are distinguishable from each other by differential mass-modification; (b) cleaving the target nucleic acid at the flanking cleavable sites; (c) denaturing the cleaved target nucleic acid to generate single-stranded plus and minus strands; (d) simultaneously cleaving the denatured plus and minus strands from a first end to a second end with an exonuclease to produce multiple sets of nested nucleic acid fragments, wherein the nested nucleic acid fragments derived from the plus strand are mass-differentiated from the nested nucleic acid fragments derived from the minus strand; (e) identifying each of the nested nucleic acid fragments produced in step (d) by mass spectrometry; and (f) determining the sequence of the target nucleic acid from the identified nested nucleic acid fragments. - View Dependent Claims (44)
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Specification