Compositions and methods for polynucleotide sequence determination
First Claim
1. A composition for identifying a nucleotide at a predetermined position of a target polynucleotide in a sample, said composition comprising:
- (a) an oligonucleotide primer comprising a first sequence which hybridizes to said target polynucleotide immediately 3′
of said nucleotide, and a second sequence which does not hybridize to said target polynucleotide in the presence of a third sequence; and
(b) an oligonucleotide probe comprising said third sequence which hybridizes to said second sequence of said oligonucleotide primer, said oligonucleotide probe labeled with a first member of a pair of interactive labels.
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Abstract
The present invention relates to a method for identifying a nucleotide at a predetermined location on a target polynucleotide. The method involves single nucleotide extension reaction comprising an oligonucleotide primer comprising a first sequence and a second sequence or a tag. The method may further comprises a probe which hybridizes to the second sequence or an anti-tag molecule which interacts with the tag, where the hybridization or interaction causes a detectable signal transfer which is indicative of the identity of the nucleotide base at the predetermined location. The invention further provides compositions and kits for performing the subject method of the invention.
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Citations
41 Claims
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1. A composition for identifying a nucleotide at a predetermined position of a target polynucleotide in a sample, said composition comprising:
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(a) an oligonucleotide primer comprising a first sequence which hybridizes to said target polynucleotide immediately 3′
of said nucleotide, and a second sequence which does not hybridize to said target polynucleotide in the presence of a third sequence; and
(b) an oligonucleotide probe comprising said third sequence which hybridizes to said second sequence of said oligonucleotide primer, said oligonucleotide probe labeled with a first member of a pair of interactive labels. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 17, 18, 19)
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12. A composition for identifying a nucleotide at a predetermined position of a target polynucleotide in a sample, said composition comprising:
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(a) an oligonucleotide primer comprising a first sequence which hybridizes to the target polynucleotide immediately 3′
of said nucleotide, and is covalently attached to a tag molecule; and
(b) an anti-tag molecule which binds to said tag molecule, said anti-tag molecule labeled with a first member of a pair of interactive labels. - View Dependent Claims (13, 14, 15, 16)
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20. A kit for identifying a nucleotide at a predetermined position of a target polynucleotide in a sample, said kit comprising:
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(a) an oligonucleotide primer comprising a first sequence which hybridizes to said target polynucleotide immediately 3′
of said nucleotide, and a second sequence which does not hybridize to said target polynucleotide in the presence of a third sequence;
(b) an oligonucleotide probe comprising said third sequence which hybridizes to said second sequence of said oligonucleotide primer, said oligonucleotide probe labeled with a first member of a pair of interactive labels; and
(c) packaging materials therefore. - View Dependent Claims (21, 22, 23, 24, 25)
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26. A kit for identifying a nucleotide at a predetermined position of a target polynucleotide in a sample, said kit comprising:
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(a) an oligonucleotide primer comprising a first sequence which hybridizes to the target polynucleotide immediately 3′
of said nucleotide, and is covalently attached to a tag molecule;
(b) an anti-tag molecule which binds to said tag molecule, said anti-tag molecule being labeled with a first member of a pair of interactive labels; and
(c) packaging materials therefore. - View Dependent Claims (27, 28, 29)
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30. A method of identifying the presence of a nucleotide at a predetermined position of a target polynucleotide, said method comprising:
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(a) incubating said target polynucleotide in a reaction mixture comprising an oligonucleotide primer comprising a first sequence which hybridizes to said target polynueleotide immediately 3′
of said nucleotide and a second sequence which does not hybridize to said target polynucleotide in the presence of a third sequence, an oligonuolcotide probe comprising said third sequence which hybridizes to said second sequence of said oligonucleotide primer, said oligonucleotide probe labeled with a first member of a pair of interactive labels, a polynucleotide chain terminator labeled with a second member of said pair of interactive labels, wherein said incubating permits said polynucleotide chain terminator to be incorporated into said oligonucleotide primer, and permits said oligonucleotide probe to hybridize to said oligonucleotide primet to permit said pair of interactive labels to generate a signal; and
(b) detecting said signal, wherein said detection is indicative of the presence of said nucleotide in said target polynucleotide. - View Dependent Claims (32, 33, 34, 35, 36, 37, 38, 39, 40, 41)
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31. A method of identifying the presence of a nucleotide at a predetermined position of a target polynuclceotide, said method comprising the steps:
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(a) incubating said target polynucleotide in a reaction mixture comprising an oligonucleotide primer comprising a first sequence which hybridizes to said target polynucleotide immediately 3′
of said nucleotide and a second sequence which does not hybridize to said target polynucleotide in the presence of a third sequence and a polynucleotide chain terminator labeled with a second member of a pair of interactive labels, wherein said incubating permits said polynucleotide chain terminator to be incorporated into said oligonucleotide primer;
(b) inctibating the oligonucleotide primer comprising said second member of said pair of interactive labels with an oligonucleotide probe comprising said third sequence which hybridizes to said second sequence of said oligonucleotide primer and said probe labeled with a first member of said pair of interactive labels, such chat formation of a hybrid between said oligonucleotide probe and said primer permits said pair of interactive labels to a generate a signal; and
(c) detecting said signal, wherein said detection is indicative of the presence of said nucleotide in said target polynucleotide.
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Specification