Methods for the isothermal amplification of nucleic acid molecules
AssigneeReplicon, Inc.
InventorJeffrey I. Auerbach
Life of this patent
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Abstract
Methods for amplifying a nucleic acid molecule which employs a single primer, and in which the amplification is performed under isothermal conditions. The invention also includes kits containing reagents for conducting the method.
Claims26
A method for amplifying a target polynucleotide region of an initial linear double-stranded nucleic acid molecule present in a sample, wherein said target polynucleotide region is flanked by recombinational sites, and wherein in said method, said linear double-stranded nucleic acid molecules are incubated in the presence of a polymerase, provided nucleotides, a site-specific recombinase, an amplification primer and a restriction endonuclease, wherein:
- said amplification primer comprises from 3' terminus to 5' terminus:
- (a) a first polynucleotide region complementary to a 3' terminal portion of said target polynucleotide region;
- (b) a second polynucleotide region, being non-complementary to any portion of said target polynucleotide region, and containing at least one modified nucleotide residue, wherein, if said second polynucleotide region were hybridized to a complementary polynucleotide, a double-stranded polynucleotide would thereby be formed that would contain one or more restriction endonuclease cleavage sites that would be recognized by said restriction endonuclease;
- and
- (c) a third polynucleotide region, which is complementary to a strand of one of said recombinational site, and which, if hybridized to a complementary polynucleotide, a double-stranded polynucleotide would thereby be formed that would contain a recombinational site recognized as a substrate by said recombinase;
- said restriction endonuclease is substantially incapable of cleaving a strand of a nucleic acid molecule that contains said modified nucleotide residue;
- said site-specific recombinase recognizes said recombinational sites, and;
- said provided nucleotides lack the modification of the modified nucleotide present in said amplification primer;
- and
- wherein said method comprises employing said recombinase to circularize said linear double-stranded nucleic acid molecules, employing said restriction endonuclease to cleave one strand of said circularized molecule to thereby form a terminus that is extendible by a polymerase, employing said polymerase to extend said formed terminus to thereby amplify one strand of said target polynucleotide, and employing said amplified strand as a template for primer extension of said amplification primer, to accomplish the amplification of said target polynucleotide.
A double-stranded circular DNA molecule comprising a recombinational site recognized as a substrate by a site-specific recombinase, a hemi-modified restriction site and a mammalian gene or a polynucleotide fragment of at least about 15 nucleotides in length of a mammalian gene.
A double-stranded linear DNA molecule comprising a lox site at each end, in direct repeat orientation, a hemi-modified restriction site and a mammalian gene or a polynucleotide fragment of at least about 15 nucleotides in length of a mammalian gene.
An oligonucleotide comprising, from 3' terminus to 5' terminus:
- (1) a first polynucleotide region complementary to a 3' terminal portion of a target polynucleotide region;
- (2) a second polynucleotide region, being non-complementary to any portion of said target polynucleotide region, and containing at least one modified nucleotide residue, wherein, if said second polynucleotide region were hybridized to a complementary polynucleotide, a double-stranded polynucleotide would thereby be formed that would contain one or more restriction endonuclease cleavage sites that would be recognized by a restriction endonuclease that is substantially incapable of cleaving a strand of a nucleic acid molecule that contains said modified nucleotide residue;
- and
- (3) a third polynucleotide region, wherein, if said third polynucleotide region were hybridized to a complementary polynucleotide, a double-stranded polynucleotide would thereby be formed that would contain a recombinational site recognized as a substrate by a site-specific recombinase.
A method for amplifying a target polynucleotide region of a double-stranded nucleic acid molecule present in a sample, said method comprising the steps:
- (A) incubating said sample under conditions sufficient to separate the strands of said double-stranded molecule and thereby form single-stranded nucleic acid molecules;
- and
- (B) incubating said formed single-stranded nucleic acid molecules in the presence of a polymerase, provided nucleotides, a restriction endonuclease, a site-specific recombinase and (i) amplification primer molecules or (ii) amplification primer molecules and target primer molecules;
- wherein:
- said amplification primer molecules are single-stranded nucleic acid molecules, comprising from 3' terminus to 5' terminus:
- (1) a first polynucleotide region complementary to a 3' terminal portion of said target polynucleotide region;
- (2) a second polynucleotide region, being non-complementary to any portion of said target polynucleotide region, and containing at least one modified nucleotide residue, wherein, if said second polynucleotide region were hybridized to a complementary polynucleotide, a double-stranded polynucleotide would thereby be formed that would contain one or more restriction endonuclease cleavage sites that would be recognized by said restriction endonuclease;
- and
- (3) a third polynucleotide region, wherein, if said third polynucleotide region were hybridized to a complementary polynucleotide, a double-stranded polynucleotide would thereby be formed that would contain a recombinational site;
- said target primer molecules are single-stranded nucleic acid molecules, comprising from 3' terminus to 5' terminus:
- (1) a first polynucleotide region having the sequence of a 5' terminal portion of said target polynucleotide;
- and
- (2) a second polynucleotide region, wherein, if said second polynucleotide region were hybridized to a complementary polynucleotide, a double-stranded polynucleotide would thereby be formed that would contain a recombinational site;
- said restriction endonuclease is substantially incapable of cleaving a strand of a nucleic acid molecule that contains said modified nucleotide residue;
- said site-specific recombinase recognizes said recombinational sites, and;
- said provided nucleotides lack the modification of the modified nucleotide present in said amplification primer;
- wherein said incubations result in the formation of an initial linear double-stranded nucleic acid molecule in which said target polynucleotide region is flanked at one end by said recombinational site;
- (c) adapting site molecule to contain a second recombinational site;
- and wherein said method comprises employing said recombinase to circularize said linear double-stranded nucleic acid molecules, employing said restriction endonuclease to cleave one strand of said circularized molecule to thereby form a terminus that is extendible by a polymerase, employing said polymerase to extend said formed terminus to thereby amplify one strand of said target polynucleotide, and employing said amplified strand as a template for primer extension of said amplification primer, to accomplish the amplification of said target polynucleotide.
Dependencies are inferred from the claim text.