Primers and methods for the detection and discrimination of nucleic acids
First Claim
Patent Images
1. A method for the quantitation or detection of one or more target nucleic acid molecules in a sample during nucleic acid synthesis comprising:
- mixing one or more a target nucleic acid molecules with one or more fluorescently labeled oligonucleotides, wherein said one or more oligonucleotides are labeled with only a single type of fluorescent label and said oligonucleotide undergoes a detectable change in fluorescence upon hybridization of said one or more oligonucleotides to said one or more target nucleic acid molecules;
incubating said mixture under conditions sufficient to synthesize one or more nucleic acid molecules complementary to all or a portion of said one or more target nucleic acid molecules, said one or more synthesized nucleic acid molecules comprising said one or more oligonucleotides; and
detecting the presence or absence or quantifying the amount of said one or more synthesized nucleic acid molecules by measuring said fluorescent label;
wherein said fluorescent label is selected from the group consisting of JOE (2′
7′
-dimethoxy-4′
5′
-dichloro-6-carboxyfluorescein), FAM (5-carboxyfluorescein), TAMRA (N,N,N′
,N′
-tetramethyl-6-carboxyrhodamine), or ROX (6-carboxy-X-rhodamine).
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Abstract
The present invention provides novel primers and methods for the detection of specific nucleic acid sequences. The primers and methods of the invention are useful in a wide variety of molecular biology applications and are particularly useful in allele specific PCR.
129 Citations
13 Claims
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1. A method for the quantitation or detection of one or more target nucleic acid molecules in a sample during nucleic acid synthesis comprising:
-
mixing one or more a target nucleic acid molecules with one or more fluorescently labeled oligonucleotides, wherein said one or more oligonucleotides are labeled with only a single type of fluorescent label and said oligonucleotide undergoes a detectable change in fluorescence upon hybridization of said one or more oligonucleotides to said one or more target nucleic acid molecules; incubating said mixture under conditions sufficient to synthesize one or more nucleic acid molecules complementary to all or a portion of said one or more target nucleic acid molecules, said one or more synthesized nucleic acid molecules comprising said one or more oligonucleotides; and detecting the presence or absence or quantifying the amount of said one or more synthesized nucleic acid molecules by measuring said fluorescent label;
wherein said fluorescent label is selected from the group consisting of JOE (2′
7′
-dimethoxy-4′
5′
-dichloro-6-carboxyfluorescein), FAM (5-carboxyfluorescein), TAMRA (N,N,N′
,N′
-tetramethyl-6-carboxyrhodamine), or ROX (6-carboxy-X-rhodamine). - View Dependent Claims (3, 5, 9, 10, 11, 12, 13)
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2. A method for quantitation or detection of one or more target nucleic acid molecules in a sample during nucleic acid amplification comprising:
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mixing one or more target nucleic acid molecules with one or more fluorescently labeled oligonucleotides under conditions sufficient to amplify one or more nucleic acid molecules complementary to all or a portion of said one or more target nucleic acid molecules, said one or more amplified nucleic acid molecules comprising said one or more oligonucleotides, wherein said one or more oligonucleotides are labeled with only a single type of fluorescent label and said oligonucleotide undergoes a detectable change in fluorescence upon hybridization of said one or more oligonucleotides to said one or more target nucleic acid molecules; and detecting the presence or absence or quantifying the amount of said one or more target nucleic acid molecules by measuring said fluorescent label;
wherein said fluorescent label is selected from the group consisting of (2′
7′
-dimethoxy-4′
5′
-dichloro-6-carboxyfluorescein), FAM (5-carboxyfluorescein), TAMRA (N,N,N′
,N′
-tetramethyl-6-carboxyrhodamine), or ROX (6-carboxy-X-rhodamine). - View Dependent Claims (4)
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6. A method for amplifying a double stranded nucleic acid molecule, comprising:
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providing a first and second primer, wherein said first primer is complementary to a sequence within or at or near the 3″
-termini of the first strand of said nucleic acid molecule and said second primer is complementary to a sequence within or at or near the 3″
-termini of the second strand of said nucleic acid molecule;hybridizing said first primer to said first strand and said second primer to said second strand in the presence of one or more polymerases, under conditions such that said primers are extended to result in the synthesis of a third nucleic acid molecule complementary to all or a portion of said first strand and a fourth nucleic acid molecule complementary to all or a portion said second strand; denaturing said first and third strands, and said second and fourth strands; and repeating the above steps one or more times, wherein one or both of said first and second primers are labeled with only a single type of fluorescent label, wherein said fluorescent label is selected from the group consisting of (2′
7′
-dimethoxy-4′
5′
-dichloro-6-carboxyfluorescein), FAM (5-carboxyfluorescein), TAMRA (N N,N′
,N′
-tetramethyl-6-carboxyrhodamine), or ROX (6-carboxy-X-rhodamine);and wherein said primer undergoes a detectable change in fluorescence upon hybridization of said one or more labeled primers to said nucleic acid molecule. - View Dependent Claims (7, 8)
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Specification