PRIMERS AND METHODS FOR THE DETECTION AND DISCRIMINATION OF NUCLEIC ACIDS
First Claim
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1. A composition for quantifying or detecting one or more target nucleic acid molecules in a sample comprising one or more detectably labeled oligonucleotides and one or more target nucleic acid molecules to be detected or quantified, wherein said oligonucleotides comprise one or more detectable labels located internally and/or at or near the 3′
- and/or 5′
termini of said oligonucleotides and wherein said label undergoes a detectable change in an observable property upon becoming part of a double stranded molecule.
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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
55 Claims
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1. A composition for quantifying or detecting one or more target nucleic acid molecules in a sample comprising one or more detectably labeled oligonucleotides and one or more target nucleic acid molecules to be detected or quantified, wherein said oligonucleotides comprise one or more detectable labels located internally and/or at or near the 3′
- and/or 5′
termini of said oligonucleotides and wherein said label undergoes a detectable change in an observable property upon becoming part of a double stranded molecule. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 41, 42, 43, 44, 45, 46)
- and/or 5′
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18. 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 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 of the 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; denaturing said first and third strand, and said second and fourth strands; and repeating the above steps one or more times, wherein one or more of the primers comprise a detectable label internally and/or at or near its 3′ and
/or 5′
termini and/or comprises one or more hairpin structures. - View Dependent Claims (19)
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20. A method for the quantification or detection of nucleic acids molecules comprising:
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mixing one or more labeled oligonucleotides with one or more nucleic acid molecules to be detected or quantitated; and detecting or measuring an increase in fluorescence associated with said oligonucleotide hybridizing to said nucleic acid molecules. - View Dependent Claims (21, 22)
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- 23. A composition comprising one or more nucleic acid molecules and at least one oligonucleotide, wherein at least a portion of said oligonucleotide is capable of hybridizing with at least a portion of said nucleic acid molecule and wherein said oligonucleotide comprises a specificity enhancing group.
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31. A method of making a composition, comprising the steps of
providing at least one oligonucleotide; - and
contacting said oligonucleotide with at least one nucleic acid molecule, wherein at least a portion of said oligonucleotide is capable of hybridizing with at least a portion of said nucleic acid molecule and wherein said oligonucleotide comprises a specificity enhancing group. - View Dependent Claims (32)
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33. A method of determining the presence of one or more particular nucleotides at a specific position or positions in a target nucleic acid molecule, comprising:
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contacting at least one target nucleic acid molecule having one or more nucleotides of interest at a specific position or positions on a target nucleic acid molecule with at least one oligonucleotide, wherein at least a portion of the oligonucleotide is capable of forming base pairs or hybridizing with at least a portion of the target nucleic acid molecule and wherein the oligonucleotide comprises at least one specificity enhancing group and/or one or more hairpin structures; and incubating the oligonucleotide and the target nucleic acid molecule under conditions sufficient to cause extension of the oligonucleotide when the 3′
-most nucleotide or nucleotides of the oligonucleotide base pair with the nucleotide or nucleotides at the specific position or positions of the target nucleic acid molecule, wherein the production of an extension product indicates the presence of the particular nucleotide at the specific position. - View Dependent Claims (34, 37, 39)
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35. A method of determining the absence of one or more particular nucleotides at a specific position or positions in a target nucleic acid molecule, comprising:
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contacting at least one target nucleic acid molecule having one or more nucleotides of interest at a specific position or positions on the target nucleic acid molecule with at least one oligonucleotide, wherein at least one portion of the oligonucleotide is capable of forming base pairs or hybridizing with at least a portion of the target nucleic acid molecule and wherein the oligonucleotide comprises at least one specificity enhancing group and/or one or more hairpin structures; and incubating the oligonucleotide and target nucleic acid molecule under conditions sufficient to inhibit or prevent extension of the oligonucleotide when the 3′
-most nucleotide or nucleotides of the oligonucleotide does not substantially base pair with the nucleotide or nucleotides of the specific position or positions of the target nucleic acid molecule, wherein the lack of or reduced production of an extension product indicates the absence of the particular nucleotide at the specific position. - View Dependent Claims (38, 40)
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36. A method of determining the presence or absence of one or more particular nucleotides at a specific position or positions in a target nucleic acid molecule, comprising:
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contacting at least first oligonucleotide with at least one target nucleic acid molecule under conditions sufficient to cause extension of the first oligonucleotide when the 3′
-most nucleotide or nucleotides of the oligonucleotide base pairs with the nucleotide or nucleotides at the specific position or positions of the target nucleic acid molecule, wherein said first oligonucleotide comprises at least one specificity enhancing group and/or at least one hairpin structure;contacting at least a second oligonucleotide with at least one target nucleic acid molecule under conditions sufficient to inhibit or prevent extension of the oligonucleotide when the 3′
-most nucleotide or nucleotides of the oligonucleotide do not substantially base pair with the nucleotide or nucleotides at the specific position or positions of the target nucleic acid molecule, wherein said second oligonucleotide comprises at least one specificity enhancing group and/or at least one hairpin structure; andcomparing the level of extension or the amount of extension product accomplished with the first oligonucleotide compared to the second oligonucleotide.
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47. A method for detecting a target nucleic acid sequence, comprising:
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contacting a sample containing a mixture of nucleic acid molecules with at least one oligonucleotide, the oligonucleotide capable of hybridizing with a target nucleic acid molecule and comprises a detectable moiety, wherein the detectable moiety undergoes a change in one or more observable property upon hybridization to the target nucleic acid molecule; and observing the observable property, wherein a change in the observable property indicates the presence of the target nucleic acid sequence.
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48. A method of determining the presence or absence of at least one particular nucleotide of interest at a specific position in a target nucleic acid molecule, comprising:
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providing at least one target nucleic acid molecule having said nucleotide of interest at a specific position; contacting said target nucleic acid molecule with at least one oligonucleotide, wherein at least a portion of the oligonucleotide is capable of forming base pairs or hybridizing with at least a portion of the nucleic acid molecule and wherein the oligonucleotide comprises at least one specificity enhancing group and/or at least one label; and contacting the oligonucleotide and the target nucleic acid molecule with a polymerase less able to extend the oligonucleotide when the 3′
-most nucleotide of the oligonucleotide does not base pair with the target nucleic acid and more able to extend the oligonucleotide when the 3′
-most nucleotide of the oligonucleotide base pairs with the target nucleic acid molecule. - View Dependent Claims (49, 50, 51, 52, 53, 54)
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55. A method for synthesizing or amplifying one or more nucleic acid molecules comprising:
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mixing one or more nucleic acid templates or targets with one or more oligonucleotides, wherein said one or more of said oligonucleotides comprises at least one hairpin structure; and incubating said mixture under conditions sufficient to synthesize or amplify one or more nucleic acid molecules complementary to all or a portion of said templates or targets.
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Specification