Method of DNA sequencing using cleavable tags
First Claim
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1. A method of determining a nucleic acid sequence, comprising:
- (a) hybridizing an oligonucleotide to a single stranded DNA within a chamber, wherein a filtration membrane is attached to the chamber, wherein the oligonucleotide is complementary to at least a portion of the single stranded DNA, and wherein the single stranded DNA is retained within the chamber by the filtration membrane;
(b) providing a DNA polymerase and four deoxynucleotide triphosphates (dNTPs) comprising dATP, dGTP, dCTP, and dTTP, wherein each dNTP is 3′
-end labeled (cdNTP) with a cleavable tag that distinguishes it from the other deoxynucleotide triphosphates;
(c) extending the single stranded DNA hybridized to the oligonucleotide by one complementary end-labeled cdNTP in a polymerase extension reaction, wherein the tag on the extended cdNTP blocks further extension by the DNA polymerase;
(d) washing the cdNTPs that are not extended onto the single stranded DNA through the filtration membrane, wherein the filtration membrane allows cdNTPs to pass through the membrane and retains the single stranded DNA in the chamber;
(e) cleaving the tag from the complementary cdNTP;
(f) washing the cleaved tag through the filtration membrane; and
(g) detecting the cleaved tag that is washed through the filtration membrane of step (f), thereby identifying the complementary dNTP.
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Abstract
The present invention provides novel systems for sequencing nucleic acid molecules using dNTPs that are 3′ end labeled with cleavable tags that block further extension and uniquely identify the bases to which they are attached. Removal of the tags liberates the 3′ ends of the extension products for further extension. In related embodiments, oligonucleotides containing sequence-related cleavable tags are employed in a ligation reaction to determine the sequence of a particular DNA sample.
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Citations
16 Claims
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1. A method of determining a nucleic acid sequence, comprising:
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(a) hybridizing an oligonucleotide to a single stranded DNA within a chamber, wherein a filtration membrane is attached to the chamber, wherein the oligonucleotide is complementary to at least a portion of the single stranded DNA, and wherein the single stranded DNA is retained within the chamber by the filtration membrane;
(b) providing a DNA polymerase and four deoxynucleotide triphosphates (dNTPs) comprising dATP, dGTP, dCTP, and dTTP, wherein each dNTP is 3′
-end labeled (cdNTP) with a cleavable tag that distinguishes it from the other deoxynucleotide triphosphates;
(c) extending the single stranded DNA hybridized to the oligonucleotide by one complementary end-labeled cdNTP in a polymerase extension reaction, wherein the tag on the extended cdNTP blocks further extension by the DNA polymerase;
(d) washing the cdNTPs that are not extended onto the single stranded DNA through the filtration membrane, wherein the filtration membrane allows cdNTPs to pass through the membrane and retains the single stranded DNA in the chamber;
(e) cleaving the tag from the complementary cdNTP;
(f) washing the cleaved tag through the filtration membrane; and
(g) detecting the cleaved tag that is washed through the filtration membrane of step (f), thereby identifying the complementary dNTP. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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9. A method of determining a nucleic acid sequence, comprising:
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(a) hybridizing a complementary oligonucleotide to a single stranded DNA, wherein the single stranded DNA is retained within a chamber by a filtration membrane that is attached to the chamber, wherein the single stranded DNA is not attached to the filtration membrane, wherein the complementary oligonucleotide is hybridized adjacent to a primer that is hybridized to the single stranded DNA, wherein the complementary oligonucleotide is 3′
-end labeled with at least one cleavable tag that distinguishes it from other complementary oligonucleotides;
(b) ligating the hybridized complementary oligonucleotide to the adjacent primer to generate a ligated complementary oligonucleotide;
(c) cleaving the tag from the ligated complementary oligonucleotide;
(d) washing the cleaved tag through the filtration membrane that allows the cleaved tag to pass through the membrane; and
(e) detecting the cleaved tag that distinguishes the complementary oligonucleotide that is washed through the filtration membrane of step (e). - View Dependent Claims (10, 11, 12, 13, 14, 15, 16)
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Specification