Use of predetermined nucleotides having altered base pairing characteristics in the amplification of nucleic acid molecules
AssigneeInvitrogen Corporation
InventorsDavid M. Schuster, Ayoub Rashtchian
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Abstract
The present invention provides improved methods for amplifying a nucleic acid molecule. More specifically, the invention provides methods for nucleic acid amplification which use primers having equivalent priming efficiency.
Claims24
A method for amplifying the concentration of a nucleic acid molecule using two primers, comprising the steps:
- (a) performing the template-dependent extension of a first primer, said primer being hybridized to a first strand of said molecule, wherein said extension forms a second strand of a nucleic acid molecule complementary to said first strand;
- (b) performing the template-dependent extension of said second strand, by extending a second primer, said primer being hybridized to said second strand of said molecule, wherein said extension forms a copy of said first strand of said nucleic acid molecule;
- (c) performing the template-dependent extension of said copy of said first strand, to thereby form a copy of said second strand of said nucleic acid molecule;
- (d) repeating steps (a), (b), and (c), to thereby achieve said amplification of said nucleic acid molecule;
- wherein at least one of said first and second primers contains;
- at least one deoxyinosine residue, and wherein said first and second primers have equivalent efficiency of primer extension.
A kit for amplifying a nucleic acid molecule containing:
- a first container containing a primer, said primer being capable of hybridizing to said nucleic acid molecule, and containing at least one deoxyinosine residue;
- and
- a second container containing an enzyme capable of adding a C nucleotide to said nucleic acid molecule, said C nucleotide being capable of base pairing with said deoxyinosine residue of said primer.
A kit for amplifying a nucleic acid molecule containing:
- a first container containing a first primer, said primer being capable of hybridizing to said nucleic acid molecule, and containing at least one deoxyinosine residue;
- and
- a second container containing a second primer;
- wherein template-dependent extension of said first primer produces a second nucleic acid molecule which is capable of hybridizing to said second primer, and wherein template-dependent extension of said second primer produces a copy of said first nucleic acid molecule.
A method for adjusting the hybridization efficiency of an oligonucleotide having a pre-determined sequence complementary to a target nucleic acid molecule, which comprises:
- A) employing as said oligonucleotide an oligonucleotide wherein at least one residue is a deoxyinosine residue;
- B) permitting said oligonucleotide to hybridize with said target molecule,
- wherein the presence of said deoxyinosine residue effects said adjustment of said hybridization efficiency.
Dependencies are inferred from the claim text.