Primers and methods for the detection and discrimination of nucleic acids
AssigneeInvitrogen Corporation
InventorsIrina Nazarenko, Ayoub Rashtchian, Joseph Solus, Richard M. Pires, Marlene Darfler, Gulilat Gebeyehu, Mekbib Astatke
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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.
Claims74
A composition for quantifying or detecting one or more target nucleic acid molecules in a sample, comprising at least three oligonucleotides, wherein the first oligonucleotide has a tail at the 5′-end of the gene-specific target primer which is non-complementary to the target sequence and which is identical to the 3′-end sequence of the second oligonucleotide;
- the second oligonucleotide is at least partially identical to the tail of the first oligonucleotide and is labeled with a fluorescent moiety;
- and the third oligonucleotide is a primer complementary to the 3′-end of the target.
A method for amplifying a double-stranded nucleic cid molecule, comprising at least three oligonucleotides, wherein the first oligonucleotide has a tail at the 5′-end of the gene-specific target primer which is non-complementary to the target sequence and which is identical to the 3′-end sequence of the second oligonucleotide;
- the second oligonucleotide is at least partially identical to the tail of the first oligonucleotide and is labeled with a fluorescent moiety;
- and the third oligonucleotide is a PCR primer complementary to the 3′-end of the target.
A method for synthesizing or amplifying one or more nucleic acid molecules comprising:
- 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 modified oligonucleotide;
- 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.
A method for synthesizing or amplifying one or more nucleic acid molecules, wherein the specificity of the nucleic acid synthesis or amplification is increased, comprising:
- 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, wherein the synthesis or amplification has increased specificity when compared to amplification or synthesis conducted with an oligonucleotide not in a hairpin conformation.
A method for synthesizing or amplifying one or more nucleic acid molecules, wherein the specificity of the nucleic acid synthesis or amplification is increased, comprising:
- 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 modified oligonucleotide;
- 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, wherein the synthesis or amplification has increased specificity when compared to amplification or synthesis conducted with an unmodified oligonucleotide.
A method for synthesizing or amplifying one or more nucleic acid molecules, wherein the synthesis or amplification inhibits or reduces mis-priming, comprising:
- 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, wherein the synthesis or amplification inhibits or reduces mis-priming when compared to amplificaiton or synthesis conducted with an oligonucleotide not in a hairpin conformation.
A method for synthesizing or amplifying one or more nucleic acid molecules, wherein the synthesis or amplification inhibits or reduces mis-priming, comprising:
- 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 modified oligonucleotide;
- 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, wherein the synthesis or amplification inhibits or reduces mis-priming when compared to amplification or synthesis conducted with an unmodified oligonucleotide.
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 modified nucleotide at or near the 3′-terminal nucleotide.
A method for amplifying a double-stranded nucleic acid molecule, comprising:
- 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 modified nucleotide at or near the 3′-terminal nucleotide.
A method of determining the presence of at least one nucleotide of interest at a specific position in a target nucleic acid molecule, comprising:
- contacting at least one target nucleic acid molecule having said nucleotide of interest at a specific position 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 a modified nucleotide at or near the 3′-terminal nucleotide;
- and
- incubating the oligonucleotide and the target nucleic acid molecule under conditions sufficient to cause extension of the oligonucleotide when the 3′-most nucleotide of the oligonucleotide base pair with the nucleotide at the specific position of the target nucleic acid molecule, wherein the presence of or increased production of an extension product indicates the presence of the particular nucleotide at the specific position.
A method of determining the absence of at least one nucleotide at a specific position in a target nucleic acid molecule, comprising:
- contacting at least one target nucleic acid molecule having said nucleotide of interest at a specific position 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 a modified nucleotide at or near the 3′-terminal nucleotide;
- 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 of the oligonucleotide does not substantially base pair with the nucleotide of the specific position of the target nucleic acid molecule, wherein the substantial reduction or no production of an extension product indicates the absence of the particular nucleotide at the specific position.
A method of determining the presence or absence of a nucleotide at a specific position in a target nucleic acid molecule, comprising:
- 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 of the oligonucleotide base pairs with the nucleotide at the specific position of the target nucleic acid molecule, wherein said first oligonucleotide comprises a modified nucleotide at or near the 3′-terminal nucleotide;
- 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 of the oligonucleotide do not substantially base pair with the nucleotide at the specific position of the target nucleic acid molecule, wherein said second oligonucleotide comprises a modified nucleotide at or near the 3′-terminal nucleotide;
- and
- comparing the level of extension or the amount of extension product accomplished with the first oligonucleotide compared to the second oligonucleotide.
A method for synthesizing or amplifying one or more nucleic acid molecules comprising:
- mixing one or more nucleic acid templates or targets with one or more oligonucleotides, wherein said one or more of said oligonucleotides comprise a modified nucleotide at or near the 3′-terminal nucleotide;
- 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.
A method for synthesizing or amplifying one or more nucleic acid molecules, wherein the specificity of the nucleic acid synthesis or amplification is increased, comprising:
- mixing one or more nucleic acid templates or targets with one or more oligonucleotides, wherein said one or more of said oligonucleotides comprises a modified ribonucleotide at or near the 3′-terminal nucleotide;
- 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, wherein the synthesis or amplification has increased specificity when compared to amplification or synthesis conducted with an oligonucleotide not modified with a modified nucleotide at or near the 3′-terminal nucleotide.
A nucleotide analogue having the formula:
- 3
- wherein,
- X is selected from the group consisting of —O—, —S—, —SO—, —SO2—, —Se—, C(R8R9), —N(R10R11), NR10, P(O2) and P(O)—O—R12;
- R1 is selected from the group consisting of nucleobases, hetroaromatic groups, hetrocyclic group and aryl;
- R2 is selected from the group consisting of H, alkyl, alkyloxy, alkylamino, alkylmercapto, aryl, aryloxy, carboxylic acid, carboxamide, aminoacid, hydroxyacid, peptide, sugar, hydroxy, amino and thio;
- R3 is selected from the group consisting of H, alkyl, alkyloxy, alkylamino, alkylmercapto, aryl, aryloxy, carboxylic acid, carboxamide, aminoacid, hydroxyacid, peptide, sugar, hydroxy, amino and thio;
- each R4 is selected from the group consisting of hydroxy, alkoxy, amino and thio;
- R5 is selected from the group consisting of H, alkyl, alkyloxy, alkylamino, alkylmercapto, aryl, aryloxy, carboxylic acid, carboxamide, aminoacid, hydroxyacid, peptide, sugar, hydroxy, amino and thio;
- U is selected from the group consisting of nucleobases, heteroaromatic groups, heterocyclic group and aryl;
- R6 is R2 (when U=CR8, NR10, N) or R6 is absent when U=—O—, —S—, —SO—, —SO2—, or —Se—;
- R7 is selected from the group consisting of triphosphate, diphosphate, monophosphate, phosphorotioate, oligonucleotide, nucleic acid, DNA, RNA, LNA[JW2] and PNA;
- R8 is selected from the group consisting of H, alkyl, alkyloxy, alkylamino, alkylmercapto, aryl, aryloxy, carboxylic acid, carboxamide, aminoacid, hydroxyacid, peptide, sugar, hydroxy, amino and thio;
- R9 is selected from the group consisting of H, alkyl, alkyloxy, alkylamino, alkylmercapto, aryl, aryloxy, carboxylic acid, carboxamide, aminoacid, hydroxyacid, peptide, sugar, hydroxy, amino and thio;
- R10, R11, and R12 are the same or different and are selected from the group consisting of alkyl, alkyloxy, alkylamino, alkylmercapto, aryl, aryloxy, carboxylic acid, carboxamide, aminoacid, hydroxyacid, peptide and sugar.
An oligonucleotide comprising:
- a cytosine or guanine or analog of said cytosine or guanine thereof at the 3′-termini, and
- one or more detectable labels on at least the second, third, fourth, fifth or sixth base from the 3′-termini.
An oligonucleotide comprising:
- an adenine or guanine at the 3′-termini,
- an overhanging guanine at the 5′-termini, and
- one or more detectable labels on at least the second, third, fourth, fifth or sixth base from a 3′-termini.
Dependencies are inferred from the claim text.