Method for sequencing polynucleotides
First Claim
1. A method for determining the nucleotide sequence of identical single strand DNA molecules comprising the steps of:
- (a) providing said single strand DNA molecules at their 3'"'"' end with a known leader sequence, said leader sequence forming a double stranded DNA hybrid with an oligonucleotide having a sequence complementary to said leader sequence;
(b) providing to said leader sequence said oligonucleotide having a sequence complementary;
(c) covalently attaching either the 3'"'"' end of said leader or the 5'"'"' end of said oligonucleotide to a solid support;
(d) forming a stable double stranded DNA hybrid, said hybrid comprising said oligonucleotide and said leader;
(e) exposing said hybrid to a DNA polymerase in the presence of optically-labeled derivatives of four nucleotide 5'"'"'-triphosphates of 2'"'"'-deoxyadenosine, 2'"'"'-deoxyguanosine, 2'"'"'-deoxycytidine and 2'"'"'-deoxythymidine, where said optically-labeled derivatives comprise a blocking group at the 3'"'"' portion thereof, said blocking group comprising an optical label capable of being removed to expose the 3'"'"' portion thereof, where each of said four derivatives is labeled with an optical label distinguishable by an optical detection means capable of detecting said optical label from the other three labels on the other three of said derivatives under conditions whereby said polymerase will add the complementary optically-labeled 3'"'"'-blocked nucleotide 5'"'"'-triphosphate to said oligonucleotide;
(f) washing any unused derivatives from said double stranded DNA hybrid;
(g) detecting the labeled derivative incorporated onto the double stranded DNA hybrid by said optical detection means thereby identifying the complement of said optically-labeled 3'"'"'-blocked nucleotide 5'"'"'-triphosphate added to said oligonucleotide present in said single stranded DNA molecules;
(h) removing, by chemical means or photochemical means, the optically-labeled 3'"'"'-blocking group from the derivative incorporated in step (g) to expose the OH group in the 3'"'"' position;
of the nucleotide 5'"'"'-triphosphate derivative incorporated into the double stranded DNA hybrid(i) separating the removed optically-labeled blocking group from said double stranded DNA hybrid;
(j) repeating steps (e) through (i) through a plurality of cycles until labeled nucleotide 5'"'"'-triphosphate derivatives can no longer be added to said oligonucleotide, whereby the result of each cycle identifies the next deoxynucleotide in sequence in said single stranded DNA molecules.
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Abstract
A method is provided for determining the sequence of nucleotides on a single strand DNA molecule. The single strand DNA molecule is attached to a leader oligonucleotide and its complementary strand to a solid state support. Fluorescently-labeled 3'"'"'-blocked nucleotide triphosphates, with each of the bases A, G, C, T having a different fluorescent label, are mixed with the bound DNA molecule in the presence of DNA polymerase. The DNA polymerase causes selective addition of only the complementary labeled NTP, thus identifying the next unpaired base in the unknown DNA strand. The 3'"'"'-blocking group is then removed, setting the system up for the next NTP addition and so on. The sequence is repeated until no more fluorescently-labeled NTPs can be detected as being added by the polymerase.
892 Citations
10 Claims
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1. A method for determining the nucleotide sequence of identical single strand DNA molecules comprising the steps of:
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(a) providing said single strand DNA molecules at their 3'"'"' end with a known leader sequence, said leader sequence forming a double stranded DNA hybrid with an oligonucleotide having a sequence complementary to said leader sequence; (b) providing to said leader sequence said oligonucleotide having a sequence complementary; (c) covalently attaching either the 3'"'"' end of said leader or the 5'"'"' end of said oligonucleotide to a solid support; (d) forming a stable double stranded DNA hybrid, said hybrid comprising said oligonucleotide and said leader; (e) exposing said hybrid to a DNA polymerase in the presence of optically-labeled derivatives of four nucleotide 5'"'"'-triphosphates of 2'"'"'-deoxyadenosine, 2'"'"'-deoxyguanosine, 2'"'"'-deoxycytidine and 2'"'"'-deoxythymidine, where said optically-labeled derivatives comprise a blocking group at the 3'"'"' portion thereof, said blocking group comprising an optical label capable of being removed to expose the 3'"'"' portion thereof, where each of said four derivatives is labeled with an optical label distinguishable by an optical detection means capable of detecting said optical label from the other three labels on the other three of said derivatives under conditions whereby said polymerase will add the complementary optically-labeled 3'"'"'-blocked nucleotide 5'"'"'-triphosphate to said oligonucleotide; (f) washing any unused derivatives from said double stranded DNA hybrid; (g) detecting the labeled derivative incorporated onto the double stranded DNA hybrid by said optical detection means thereby identifying the complement of said optically-labeled 3'"'"'-blocked nucleotide 5'"'"'-triphosphate added to said oligonucleotide present in said single stranded DNA molecules; (h) removing, by chemical means or photochemical means, the optically-labeled 3'"'"'-blocking group from the derivative incorporated in step (g) to expose the OH group in the 3'"'"' position;
of the nucleotide 5'"'"'-triphosphate derivative incorporated into the double stranded DNA hybrid(i) separating the removed optically-labeled blocking group from said double stranded DNA hybrid; (j) repeating steps (e) through (i) through a plurality of cycles until labeled nucleotide 5'"'"'-triphosphate derivatives can no longer be added to said oligonucleotide, whereby the result of each cycle identifies the next deoxynucleotide in sequence in said single stranded DNA molecules. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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Specification