Nucleic acid amplification
First Claim
1. A method of amplifying a target nucleic acid sequence, the method comprising, bringing into contact a set of primers, DNA polymerase, and a target sample, and incubating the target sample under conditions that promote replication of the target sequence, wherein the target sample is not subjected to denaturing conditions, wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand.
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Abstract
Disclosed are compositions and a method for amplification of nucleic acid sequences of interest. The disclosed method generally involves replication of a target sequence such that, during replication, the replicated strands are displaced from the target sequence by strand displacement replication of another replicated strand. In one form of the disclosed method, the target sample is not subjected to denaturing conditions. It was discovered that the target nucleic acids, genomic DNA, for example, need not be denatured for efficient multiple displacement amplification. The primers used can be hexamer primers. The primers can also each contain at least one modified nucleotide such that the primers are nuclease resistant. The primers can also each contain at least one modified nucleotide such that the melting temperature of the primer is altered relative to a primer of the same sequence without the modified nucleotide(s). The DNA polymerase can be φ29 DNA polymerase.
68 Citations
223 Claims
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1. A method of amplifying a target nucleic acid sequence, the method comprising,
bringing into contact a set of primers, DNA polymerase, and a target sample, and incubating the target sample under conditions that promote replication of the target sequence, wherein the target sample is not subjected to denaturing conditions, wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand.
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111. A method of amplifying a target nucleic acid sequence, the method comprising,
bringing into contact a set of primers, DNA polymerase, and a target sample, and incubating the target sample under conditions that promote replication of the target sequence, wherein nucleic acids in the target sample are not separated from other material in the target sample, wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand.
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137. A method of amplifying a target nucleic acid sequence, the method comprising,
bringing into contact a set of primers, DNA polymerase, and a target sample, and incubating the target sample under conditions that promote replication of the target sequence, wherein the target sample is a crude cell lysate, wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand.
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138. A method of amplifying a target nucleic acid sequence, the method comprising,
bringing into contact a set of primers, DNA polymerase, and a target sample, and incubating the target sample under conditions that promote replication of the target sequence, wherein the primers are 5, 6, 7, 8, 9, or 10 nucleotides long, wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand.
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143. A method of amplifying a target nucleic acid sequence, the method comprising,
bringing into contact a set of primers, DNA polymerase, and a target sample, and incubating the target sample under conditions that promote replication of the target sequence, wherein the primers each contain at least one modified nucleotide such that the primers are nuclease resistant, wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand.
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144. A method of amplifying a target nucleic acid sequence, the method comprising,
bringing into contact a set of primers, DNA polymerase, and a target sample, and incubating the target sample under conditions that promote replication of the target sequence, wherein the primer-target sample is not subjected to denaturing conditions, wherein the primers are 6 nucleotides long, wherein the primers each contain at least one modified nucleotides such that the primers are nuclease resistant, and wherein DNA polymerase is φ - 29 DNA polymerase,
wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand.
- 29 DNA polymerase,
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145. A method of amplifying a target nucleic acid sequence, the method comprising,
bringing into contact a set of primers, DNA polymerase, and a target sample, and incubating the target sample under conditions that promote replication of the target sequence, wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand, diluting the replicated strands, bringing into contact a set of primers, DNA polymerase, and the diluted replicated strands, and incubating the replicated strands under conditions that promote replication of the target sequence, wherein replication of the target sequence results in additional replicated strands, wherein during replication at least one of the additional replicated strands is displaced from the target sequence by strand displacement replication of another additional replicated strand.
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147. A method of amplifying a target nucleic acid sequence, the method comprising,
(a) mixing a set of primers with a target sample, to produce a primer-target sample mixture, and incubating the primer-target sample mixture under conditions that promote hybridization between the primers and the target sequence in the primer-target sample mixture, wherein the primer-target sample is not subjected to denaturing conditions, (b) mixing DNA polymerase with the primer-target sample mixture, to produce a polymerase-target sample mixture, and incubating the polymerase-target sample mixture under conditions that promote replication of the target sequence, wherein the set of primers comprises a right set of primers and a left set of primers, wherein the target sequence is double-stranded, having a first and a second strand, wherein the right set primers are all complementary to the first strand of the target sequence and the left set primers are all complementary to the second strand of the target sequence, wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand.
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149. A method of amplifying a target nucleic acid sequence, the method comprising,
(a) mixing a set of primers with a target sample, to produce a primer-target sample mixture, and incubating the primer-target sample mixture under conditions that promote hybridization between the primers and the target sequence in the primer-target sample mixture, wherein the primer-target sample is not subjected to denaturing conditions, (b) mixing DNA polymerase with the primer-target sample mixture, to produce a polymerase-target sample mixture, and incubating the polymerase-target sample mixture under conditions that promote replication of the target sequence, wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand, wherein the target sequence is a nucleic acid sample of substantial complexity, and wherein the set of primers comprises primers having random nucleotide sequences.
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150. A method of amplifying a target nucleic acid sequence, the method comprising,
(a) mixing a set of primers with a target sample, to produce a primer-target sample mixture, and incubating the primer-target sample mixture under conditions that promote hybridization between the primers and the target sequence in the primer-target sample mixture, wherein the primer-target sample is not subjected to denaturing conditions, (b) mixing DNA polymerase with the primer-target sample mixture, to produce a polymerase-target sample mixture, and incubating the polymerase-target sample mixture under conditions that promote replication of the target sequence, wherein all of the primers in the set of primers are complementary to the same strand in the target sequence, wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand.
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152. A method of amplifying a target nucleic acid sequence, the method comprising,
(a) mixing a set of primers with a target sample, to produce a primer-target sample mixture, and incubating the primer-target sample mixture under conditions that promote hybridization between the primers and the target sequence in the primer-target sample mixture, wherein the primer-target sample is not subjected to denaturing conditions, (b) mixing DNA polymerase with the primer-target sample mixture, to produce a polymerase-target sample mixture, and incubating the polymerase-target sample mixture under conditions that promote replication of the target sequence, wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand, wherein the target sequence is a nucleic acid sample of substantial complexity, and wherein the set of primers comprises primers having random nucleotide sequences, wherein each primer comprises a constant portion and a random portion, wherein the constant portion of each primer has the same nucleotide sequence and the random portion of each primer has a random nucleotide sequence.
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153. A method of amplifying a target nucleic acid sequence, the method comprising,
(a) mixing a set of primers with a target sample, to produce a primer-target sample mixture, and incubating the primer-target sample mixture under conditions that promote hybridization between the primers and the target sequence in the primer-target sample mixture, wherein the primer-target sample is not subjected to denaturing conditions, (b) mixing DNA polymerase with the primer-target sample mixture, to produce a polymerase-target sample mixture, and incubating the polymerase-target sample mixture under conditions that promote replication of the target sequence, wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand, wherein the conditions that promote replication of the target sequence do not involve thermal cycling, and wherein the target sequence is concatenated DNA.
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154. A method of amplifying a target nucleic acid sequence, the method comprising,
bringing into contact a set of primers, DNA polymerase, and a target sample, and incubating the target sample under conditions that promote replication of the target sequence, wherein the target sample is not subjected to denaturing conditions, wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand.
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156. A method of amplifying a target nucleic acid sequence, the method comprising,
bringing into contact a set of primers, DNA polymerase, and a target sample, and incubating the target sample under conditions that promote replication of the target sequence, wherein the target sample is not subjected to denaturing conditions, wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand, wherein the target sequence is a nucleic acid sample of substantial complexity, and wherein the set of primers comprises primers having random nucleotide sequences.
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157. A method of amplifying a target nucleic acid sequence, the method comprising,
bringing into contact a set of primers, DNA polymerase, and a target sample, and incubating the target sample under conditions that promote replication of the target sequence, wherein the target sample is not subjected to denaturing conditions, wherein all of the primers in the set of primers are complementary to the same strand in the target sequence, wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand.
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159. A method of amplifying a target nucleic acid sequence, the method comprising,
bringing into contact a set of primers, DNA polymerase, and a target sample, and incubating the target sample under conditions that promote replication of the target sequence, wherein the target sample is not subjected to denaturing conditions, wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand, wherein the target sequence is a nucleic acid sample of substantial complexity, and wherein the set of primers comprises primers having random nucleotide sequences, wherein each primer comprises a constant portion and a random portion, wherein the constant portion of each primer has the same nucleotide sequence and the random portion of each primer has a random nucleotide sequence.
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160. A method of amplifying a target nucleic acid sequence, the method comprising,
bringing into contact a set of primers, DNA polymerase, and a target sample, and incubating the target sample under conditions that promote replication of the target sequence, wherein the target sample is not subjected to denaturing conditions, wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand, wherein the conditions that promote replication of the target sequence do not involve thermal cycling, and wherein the target sequence is concatenated DNA.
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161. A method of amplifying a target nucleic acid sequence, the method comprising,
bringing into contact a set of primers, DNA polymerase, and a target sample, and incubating the target sample under conditions that promote replication of the target sequence, wherein the target sample is not subjected to heat denaturing conditions, wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand.
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162. A method of labeling nucleic acids produced by strand displacement replication, the method comprising
labeling nucleic acids produced by strand displacement replication using terminal deoxynucleotidyl transferase.
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165. A method of labeling nucleic acids produced by strand displacement replication, the method comprising
incorporating modified nucleotides into nucleic acids produced by strand displacement replication during replication.
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169. A method of attaching nucleic acids produced by strand displacement replication, the method comprising
adding modified nucleotides to the 3′ - ends of nucleic acids produced by strand displacement replication using terminal deoxynucleotidyl transferase, and
reacting the modified nucleotides with a solid-state support thereby attaching the nucleic acids to the solid-state support.
- ends of nucleic acids produced by strand displacement replication using terminal deoxynucleotidyl transferase, and
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170. A microarray comprising nucleic acids produced by strand displacement replication coupled or adhered to a solid-state substrate.
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171. A method of generating probes based on a target nucleic acid sequence, the method comprising,
bringing into contact a set of primers, DNA polymerase, and a target sample, and incubating the target sample under conditions that promote replication of the target sequence, wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand, wherein the replicated strands are used as hybridization probes.
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183. A method of amplifying messenger RNA, the method comprising,
reverse transcribing messenger RNA to produce a first strand cDNA, bringing into contact a set of primers, DNA polymerase, and the first strand cDNA, and incubating under conditions that promote replication of the first strand cDNA, wherein replication of the first strand cDNA results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the first strand cDNA by strand displacement replication of another replicated strand.
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211. A method of amplifying a target nucleic acid sequence, the method comprising,
partially degrading RNA in a target sample, bringing into contact DNA polymerase, and the target sample, and incubating the target sample under conditions that promote replication of the target sequence, wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand.
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212. A method of comparative genome hybridization, the method comprising,
hybridizing nucleic acids produced by strand displacement replication of a first sample with nucleic acids produced by strand displacement replication of a second sample.
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214. A method of amplifying a target nucleic acid sequence, the method comprising,
bringing into contact a set of primers, DNA polymerase, and a target sample, and incubating the target sample under conditions that promote replication of the target sequence, wherein a circular nucleic acid molecule comprises the target sequence, wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand.
Specification