Use of deoxyinosine containing primers to balance primer efficiency in the amplification of nucleic acid molecules
First Claim
1. An improved method for amplifying the concentration of a target nucleic acid molecule by using a first and second primer to form a double-stranded nucleic acid molecule, wherein said first primer hybridizes to a first strand of said double-stranded nucleic acid molecule, and said second primer hybridizes to a second strand of said double-stranded nucleic acid molecule, and wherein said primers exhibit different Tm s with respect to one another, when hybridized to said double-stranded nucleic acid molecule, said improved method comprising the steps:
- (a) replacing at least one residue in at least one of said primers with at least one deoxyinosine, wherein said replacement equalizes the Tm s of the first and second primers with respect to one another, when hybridized to said double-stranded nucleic acid molecule;
(b) performing the template-dependent extension of said first primer, said first primer being hybridized to a first strand of said double-stranded molecule, wherein said extension forms a second strand of nucleic acid complementary to said first strand of said double-stranded nucleic acid molecule;
(c) performing the template-dependent extension of said second strand, by extending said second primer, said second primer being hybridized to said second strand of said double-stranded molecule, wherein said extension forms a copy of said first strand of said double-stranded nucleic acid molecule;
(d) performing the template-dependent extension of said copy of said first strand, to thereby form a copy of said second strand of said double-stranded nucleic acid molecule;
(e) repeating steps (b), (c) and (d), to thereby achieve said amplification of said target nucleic acid molecule.
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Abstract
The present invention provides improved methods for amplifying a nucleic acid molecule. Specifically, the invention involves replacing at least one nucleotide of an oligonucleotide with a deoxyinosine residue, so as to more equalize the efficiency with which that oligonucloetide and a second oligonucleotide hybridize to a target molecule, and then amplifying the target molecule using, for example, the polymerase chain reaction. Improved amplification results from the improvement in the relative hybridization efficiencies.
50 Citations
16 Claims
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1. An improved method for amplifying the concentration of a target nucleic acid molecule by using a first and second primer to form a double-stranded nucleic acid molecule, wherein said first primer hybridizes to a first strand of said double-stranded nucleic acid molecule, and said second primer hybridizes to a second strand of said double-stranded nucleic acid molecule, and wherein said primers exhibit different Tm s with respect to one another, when hybridized to said double-stranded nucleic acid molecule, said improved method comprising the steps:
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(a) replacing at least one residue in at least one of said primers with at least one deoxyinosine, wherein said replacement equalizes the Tm s of the first and second primers with respect to one another, when hybridized to said double-stranded nucleic acid molecule; (b) performing the template-dependent extension of said first primer, said first primer being hybridized to a first strand of said double-stranded molecule, wherein said extension forms a second strand of nucleic acid complementary to said first strand of said double-stranded nucleic acid molecule; (c) performing the template-dependent extension of said second strand, by extending said second primer, said second primer being hybridized to said second strand of said double-stranded molecule, wherein said extension forms a copy of said first strand of said double-stranded nucleic acid molecule; (d) performing the template-dependent extension of said copy of said first strand, to thereby form a copy of said second strand of said double-stranded nucleic acid molecule; (e) repeating steps (b), (c) and (d), to thereby achieve said amplification of said target nucleic acid molecule. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. An improved method for amplifying the concentration of a target nucleic acid molecule by using a first and a second oligonucleotide primers, wherein said first and second primers hybridize to a nucleic acid molecule, such that they may be ligated to one another and thereby form said target molecule, and exhibit different Tm s with respect to one another, when hybridized to said nucleic acid molecule;
- said improved method comprising the steps;
(a) replacing at least one residue in at least one of said primers with at least one deoxyinosine, wherein said replacement equalizes the Tm s of the first and second primers with respect to one another, when hybridized to said nucleic acid molecule; (b) hybridizing said first and second primers to said nucleic acid molecule; and (c) ligating said hybridized first and second primers to one another, wherein said ligation forms said target nucleic acid molecule. - View Dependent Claims (14, 15, 16)
- said improved method comprising the steps;
Specification