US5733733A — Methods for the isothermal amplification of nucle…

Claim 1

Independent Claim 317 words

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.

Claim 2

Independent Claim 317 words

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.

Claim 3

Dependent Claim 26 words

The method of claim 1, wherein one strand of said initial linear double-stranded nucleic acid molecule comprises a primer extension product of said amplification primer molecules.

Claim 4

Dependent Claim 93 words

The method of claim 1, wherein one strand of said initial linear double-stranded nucleic acid molecule comprises a primer extension product of a target primer molecule, said target primer molecule being single-stranded, and comprising from 3' terminus to 5' terminus: (1) a first polynucleotide region having the sequence of a 5' terminal portion of said target polynucleotide region; 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 recognized by said recombinase.

Claim 5

Dependent Claim 68 words

The method of claim 2, wherein said amplification primer molecules additionally contain a fourth polynucleotide region, said fourth polynucleotide region of said amplification primer molecules being located 5' to said third polynucleotide region of said amplification primer molecules, and having a nucleotide sequence complementary to at least a portion thereof, such that said third and fourth polynucleotide regions of said amplification primer molecules are hybridized to one another.

Claim 6

Dependent Claim 68 words

The method of claim 3, wherein said target primer molecules additionally contain a third polynucleotide region, said third polynucleotide region of said target primer molecules being located 5' to said second polynucleotide region of said target primer molecules, and having a nucleotide sequence complementary to at least a portion thereof, such that said second and third polynucleotide regions of said target primer molecules are hybridized to one another.

Claim 7

Dependent Claim 103 words

The method of claim 2 wherein the strand of said initial linear double-stranded nucleic acid molecule that is not said printer extension product of said amplification primer molecules comprises a primer extension product of target primer molecules, said target primer molecules being single-stranded, and 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 recognized by said recombinase.

Claim 8

Dependent Claim 68 words

The method of claim 6, wherein said amplification primer molecules additionally contain a fourth polynucleotide region, said fourth polynucleotide region of said amplification primer molecules being located 5' to said third polynucleotide region of said amplification primer molecules, and having a nucleotide sequence complementary to at least a portion thereof, such that said third and fourth polynucleotide regions of said amplification primer molecules are hybridized to one another.

Claim 9

Dependent Claim 68 words

The method of claim 6, wherein said target primer molecules additionally contain a third polynucleotide region, said third polynucleotide region of said target primer molecules being located 5' to said second polynucleotide region of said target primer molecules, and having a nucleotide sequence complementary to at least a portion thereof, such that said second and third polynucleotide regions of said target primer molecules are hybridized to one another.

Claim 10

Dependent Claim 12 words

The method of claim 1, wherein said modified nucleotides are methylated nucleotides.

Claim 11

Dependent Claim 12 words

The method of claim 1, wherein said modified nucleotides are (α-thio)phosphorothioated nucleotides.

Claim 12

Dependent Claim 36 words

The method of claim 1, wherein said amplification primer is provided in limiting amounts, such that said method amplifies one strand of said double-stranded nucleic acid target molecule to a greater extent than the other strand.

Claim 13

Dependent Claim 41 words

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.

Claim 14

Dependent Claim 41 words

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.

Claim 15

Dependent Claim 40 words

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.

Claim 16

Dependent Claim 40 words

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.

Claim 17

Dependent Claim 143 words

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.

Claim 18

Dependent Claim 143 words

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.

Claim 19

Dependent Claim 13 words

The oligonucleotide of claim 14, wherein said target polynucleotide is a mammalian gene.

Claim 20

Independent Claim 433 words

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.

Claim 21

Independent Claim 433 words

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.

Claim 22

Dependent Claim 20 words

The method of claim 16, wherein said site-specific recombinase is Cre, and wherein said recombinational site is a lox site.

Claim 23

Dependent Claim 105 words

The method of claim 16, wherein said incubation step (B) is conducted in the presence of a polymerase and nucleotides, said incubation being under conditions sufficient to permit either: (i) said first polynucleotide region of said amplification primer molecules to hybridize to the 3' terminus of said target polynucleotide, and template dependent extension of said amplification primer molecules, to thereby form amplification primer extension products; or (ii) said first polynucleotide region of said target primer molecules to hybridize to the 3' terminus of a complement of said target polynucleotide, and template dependent extension of said target primer molecules, to thereby form target primer extension products.

Claim 24

Dependent Claim 189 words

The method of claim 18, which additionally includes the steps: (D) incubating said primer extension products (i) or (ii) under conditions sufficient to denature double-stranded nucleic acid molecules and thereby produce unhybridized primer extension products; (E) where amplification primer was employed, incubating said unhybridized amplification primer extension products in the presence of target primer molecules; where target primer was employed, incubating said unhybridized target primer extension products in the presence of amplification primer molecules; wherein said incubation is conducted in the presence of a polymerase and nucleotides, said incubation being under conditions sufficient to permit (i) said unhybridized primer extension products to hybridize to the 3' terminus of said prier molecules, and (ii) template dependent extension of said primer extension products, and of said primer molecules, to thereby form linear double-stranded nucleic acid molecules having said recombinational site at each end, and containing said target polynucleotide that is to be amplified and a hemi-modified restriction site; and (F) incubating said formed linear double-stranded nucleic acid molecules in the presence of a site-specific recombinase that recognizes said recombinational site as a substrate, said restriction endonuclease, said polymerase and said nucleotides.

Claim 25

Dependent Claim 20 words

The method of claim 19, wherein said site-specific recombinase is Cre, and wherein said recombinational site is a lox site.

Claim 26

Dependent Claim 22 words

The method of claim 20, wherein in step (B) amplification primer is employed, and wherein in step (E) target primer is employed.

Claim 27

Dependent Claim 22 words

The method of claim 20, wherein in step (B) target primer is employed, and wherein in step (E) simplification primer is employed.

Claim 28

Dependent Claim 16 words

The double-stranded circular DNA molecule of claim 12, wherein said recombinational site is a lox site.

Claim 29

Dependent Claim 13 words

The oligonucleotide of claim 14, wherein said recombinational site is a lox site.

Claim 30

Dependent Claim 20 words

The method of claim 1, wherein said site-specific recombinase is Cre, and wherein said recombinational site is a lox site.

Claim 31

Dependent Claim 20 words

The method of claim 3, wherein said site-specific recombinase is Cre, and wherein said recombinational site is a lox site.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	2001-03-30	2001-09-29	2002-03-31	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	2005-03-30	2005-09-29	2006-03-31	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2009-03-30	2009-09-29	2010-03-31	$7700.00	Window Closed / Lapsed

Methods for the isothermal amplification of nucleic acid molecules

Abstract

Inventors

Bibliographic Details

Claims Analysis

References Cited (Backward Citations)

USPTO Annuity & Maintenance Windows

Assigned Categories & Tags

Assign Category Tag