Compositions and methods for polynucleotide sequence determination
AssigneeStratagene
InventorsJoseph A. Sorge, Bahram Arezi, Holly Hogrefe
Life of this patent
Estimating the term…
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.
Claims54
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.
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 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.
A kit for identifying a nucleotide at a predetermined position of a target polynucleotide in a sample, said kit 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;
- (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.
A kit for identifying a nucleotide at a predetermined position of a target polynucleotide in a sample, said kit comprising:
- (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.
A method of identifying the presence of a nucleotide at a predetermined position of a target polynucleotide, said method comprising:
- (a) incubating said target polynucleotide in a reaction mixture comprising an oligonucleotide primer which hybridizes to said target polynucleotide immediately 3′ of said nucleotide, an oligonucleotide probe which hybridizes to said oligonucleotide primer and 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 primer 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.
A method of identifying the presence of a nucleotide at a predetermined position of a target polynucleotide, said method comprising the steps:
- (a) incubating said target polynucleotide in a reaction mixture comprising an oligonucleotide primer which hybridizes to said target polynucleotide immediately 3′ of said nucleotide 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) incubating the oligonucleotide primer comprising said second member of said pair of interactive labels with an oligonucleotide probe labeled with a first member of said pair of interactive labels, such that 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.
A method of identifying the presence of a nucleotide at a predetermined position of a target polynucleotide, said method comprising:
- (a) incubating said target polynucleotide in a reaction mixture comprising an anti-tag molecule 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, and an oligonucleotide primer which hybridizes to said target polynucleotide immediately 3′ of the nucleotide, said oligonucleotide primer covalently coupled to a tag molecule, wherein said incubating permits said polynucleotide chain terminator to be incorporated into said oligonucleotide primer, and said incubating also permits said anti-tag molecule to interact with said tag molecule on said oligonucleotide primer, so that said pair of interactive labels generate a signal;
- and
- (b) detecting said signal, wherein said detection is indicative of the presence of said nucleotide in said target polynucleotide.
A method of identifying the presence of a nucleotide at a predetermined position of a target polynucleotide, said method comprising:
- (a) incubating said target polynucleotide in a reaction mixture comprising an oligonucleotide primer which hybridizes to said target polynucleotide immediately 3′ of said nucleotide, an oligonucleotide probe which hybridizes to said oligonucleotide primer and labeled with a first member of a pair of interactive labels, a conventional deoxynucleotide labeled with a second member of said pair of interactive labels, wherein said incubating permits said labeled conventional deoxynucleotide to be incorporated into said oligonucleotide primer at a position corresponding to the predetermined position of the target polynucleotide, and permits said oligonucleotide probe to hybridize to said oligonucleotide primer 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.
A method of identifying the presence of a nucleotide at a predetermined position of a target polynucleotide, said method comprising the steps:
- (a) incubating said target polynucleotide in a reaction mixture comprising an oligonucleotide primer which hybridizes to said target polynucleotide immediately 3′ of said nucleotide and a conventional deoxynucleotide labeled with a second member of a pair of interactive labels, wherein said incubating permits said conventional deoxynucleotide to be incorporated into said oligonucleotide primer at a position corresponding to the predetermined position of the target polynucleotide;
- (b) incubating the oligonucleotide primer comprising said second member of said pair of interactive labels with an oligonucleotide probe labeled with a first member of said pair of interactive labels, such that 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.
A method of identifying the presence of a nucleotide at a predetermined position of a target polynucleotide, said method comprising:
- (a) incubating said target polynucleotide in a reaction mixture comprising an anti-tag molecule labeled with a first member of a pair of interactive labels, a conventional deoxynucleotide labeled with a second member of said pair of interactive labels, and an oligonucleotide primer which hybridizes to said target polynucleotide immediately 3′ of the nucleotide, said oligonucleotide primer covalently coupled to a tag molecule, wherein said incubating permits said conventional deoxynucleotide to be incorporated into said oligonucleotide primer at a position corresponding to the predetermined position of the target polynucleotide, and said incubating also permits said anti-tag molecule to interact with said tag molecule on said oligonucleotide primer, so that said pair of interactive labels generate a signal;
- and
- (b) detecting said signal, wherein said detection is indicative of the presence of said nucleotide in said target polynucleotide.
Dependencies are inferred from the claim text.