Primers and methods for the detection and discrimination of nucleic acids
First Claim
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1. A composition comprising one or more target nucleic acid molecules and at least two different oligonucleotides, wherein said oligonucleotides are extendable by nucleic acid synthesis and wherein said oligonucleotides, when extended by nucleic acid synthesis, are incorporated into a double-stranded nucleic acid product on opposite strands, wherein said first oligonucleotide comprises a fluorescent moiety at or near its 3′
- -terminal nucleotide and said second oligonucleotide comprises a quenching moiety at or near its 3′
-terminal nucleotide; and
wherein said first oligonucleotide exhibits a detectable change upon incorporation into said double-stranded nucleic acid product.
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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.
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Citations
16 Claims
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1. A composition comprising one or more target nucleic acid molecules and at least two different oligonucleotides, wherein said oligonucleotides are extendable by nucleic acid synthesis and wherein said oligonucleotides, when extended by nucleic acid synthesis, are incorporated into a double-stranded nucleic acid product on opposite strands, wherein said first oligonucleotide comprises a fluorescent moiety at or near its 3′
- -terminal nucleotide and said second oligonucleotide comprises a quenching moiety at or near its 3′
-terminal nucleotide; and
wherein said first oligonucleotide exhibits a detectable change upon incorporation into said double-stranded nucleic acid product. - View Dependent Claims (2, 4, 5, 6, 10, 11, 12, 13)
- -terminal nucleotide and said second oligonucleotide comprises a quenching moiety at or near its 3′
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3. 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 portion of the first strand of said nucleic molecule and said second primer is complementary to a portion 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 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 are synthesized;
wherein said first and second primers are incorporated into said third and said fourth nucleic acid molecules, respectively;denaturing said first and third strand, and said second and fourth strands; and repeating the above steps one or more times, wherein said first primer comprises a fluorescent moiety at or near its 3′
-terminal nucleotide and said second primer comprises a quencher moiety at or near its 3′
-terminal nucleotide, wherein said fluorescent moiety exhibits a detectable change upon incorporation of said first primer into said third nucleic acid molecules. - View Dependent Claims (7, 8, 9, 14, 15, 16)
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Specification