DNA sequencing by mass spectrometry via exonuclease degradation
First Claim
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1. A process for determining the sequence of a target nucleic acid, comprising the steps of:
- (i) obtaining multiple nucleic acid copies of the target nucleic acid, wherein the multiple copies contain at least one mass-modified nucleotide, corresponding to one of the four possible nucleotide bases;
(ii) cleaving the multiple nucleic acid copies from a first end to a second end with an exonuclease having an activity, which is inhibited by the mass-modified nucleotide, thereby generating base-terminated nucleic acid fragments;
(iii) identifying the nested nucleic acid fragments by mass spectrometry; and
(iv) determining the sequence of the target nucleic acid from the identified nested nucleic acid fragment.
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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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23 Claims
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1. A process for determining the sequence of a target nucleic acid, comprising the steps of:
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(i) obtaining multiple nucleic acid copies of the target nucleic acid, wherein the multiple copies contain at least one mass-modified nucleotide, corresponding to one of the four possible nucleotide bases; (ii) cleaving the multiple nucleic acid copies from a first end to a second end with an exonuclease having an activity, which is inhibited by the mass-modified nucleotide, thereby generating base-terminated nucleic acid fragments; (iii) identifying the nested nucleic acid fragments by mass spectrometry; and (iv) determining the sequence of the target nucleic acid from the identified nested nucleic acid fragment. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)
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Specification