Fingerprinting using single specific primers in low stringency polymerase chain reaction conditions
First Claim
1. A method to obtain a signature characteristic of the nucleotide sequence of a purified cloned or amplified defined DNA segment which method consists essentially of:
- subjecting said segment to multiply cycles of replication in the presence of a single primer fully complementary to a portion of at least about ten nucleotides of said segment, wherein said portion is not a repetitive sequence or part of a gene cluster within said DNA segment under conditions of low stringency at 25°
C.-40°
C. a primer concentration in the range of 2-10 μ
M, and a polymerase concentration in the range of 60-200 units/ml to obtain a mixture of replicated DNA molecules which result from said multiplicity of low stringency cycles of replication; and
determining the size of the replicated DNA molecules in the mixture thus obtaining a signature characteristic of said nucleotide sequence.
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Abstract
A straightforward method to characterize a nucleotide sequence of a defined DNA region is described. A purified segment representing the defined region is subjected to repeated cycles of amplification (PCR) using a single primer complementary to a portion of the segment under conditions of low stringency and high polymerase and primer concentrations. PCR under these conditions results in a mixture of amplified DNA molecules the composition of which is characteristic of the DNA segment assessed. Determination of the composition of the mixture can be obtained by subjecting the mixture to standard chromatographic or electrophoretic techniques to obtain a pattern of fragments which represents a "signature" of the nucleotide sequence.
8 Citations
11 Claims
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1. A method to obtain a signature characteristic of the nucleotide sequence of a purified cloned or amplified defined DNA segment which method consists essentially of:
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subjecting said segment to multiply cycles of replication in the presence of a single primer fully complementary to a portion of at least about ten nucleotides of said segment, wherein said portion is not a repetitive sequence or part of a gene cluster within said DNA segment under conditions of low stringency at 25°
C.-40°
C. a primer concentration in the range of 2-10 μ
M, and a polymerase concentration in the range of 60-200 units/ml to obtain a mixture of replicated DNA molecules which result from said multiplicity of low stringency cycles of replication; anddetermining the size of the replicated DNA molecules in the mixture thus obtaining a signature characteristic of said nucleotide sequence. - View Dependent Claims (2, 3, 4, 5, 6)
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7. A signature characteristic of a defined DNA segment which signature consists essentially of a developed chromatographic or electrophoretic support obtained by chromatography or electrophoresis of a mixture
wherein said mixture is obtained by a process consisting essentially of subjecting said DNA segment to multiple cycles of replication in the presence of a single primer fully complementary to a portion consisting of at least about 10 nucleotides of said segment, wherein said portion is not a repetitive sequence or part of a gene cluster within said DNA segment, under conditions of low stringency at 25° - C.-40°
C., a primer concentration in the range of 2-10 μ
M, and a polymerase concentration in the range of 60-200 units/ml.
- C.-40°
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8. A method to obtain a signature characteristic of the nucleotide sequence of at least two different DNA segments which method comprises
amplifying regions representing each of said at least two DNA segments from duplex DNA in a standard polymerase chain reaction (PCR) conducted in the presence of primers complementary to the opposing strands of said duplex at opposite extremities of each said DNA region, wherein one of the primers in the PCR of each region is extended at its 5'"'"' end by a DNA sequence tail common to primers for each region to obtain said at least two different DNA segments in purified and amplified form, followed by a process that consists essentially of subjecting each segment to multiple cycles of replication in the presence of a single primer fully complementary to a portion consisting of at least about 10 nucleotides of said tail, wherein said portion is not a repetitive sequence or part of a gene cluster within said DNA segment under conditions of low stringency at 25° - C.-40°
C., a primer concentration in the range of 2-10 μ
M, and a polymerase concentration in the range of 60-200 units/ml to obtain a mixture of replicated DNA molecules which result from said multiplicity of low stringency cycles of replication; anddetermining the size of the replication DNA molecules in the mixture.
- C.-40°
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9. A method to determining the presence or absence of an allele predisposing a subject to a disease or condition which method comprises:
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obtaining genomic DNA from said subjects; providing, in a purified and amplified or cloned form, a DNA segment representing a defined region of said genomic DNA from the locus of the allele;
followed by a process consisting essentially ofsubjecting said segment to multiple cycles of replication in the presence of a single primer fully complementary to a portion consisting of at least about ten nucleotides of said segment, wherein said portion is not a repetitive sequence or part of a gene cluster within said DNA segment under conditions of low stringency at 25°
C.-40°
C., a primer concentration in the range of 2-10 μ
M, and a polymerase concentration in the range of 60-200 units/ml to obtain a mixture of replicated DNA molecules which result from said multiplicity of low stringency cycles of replication; anddetermining the sizes of the replicated DNA molecules in the mixture thus obtaining a signature characteristic of said locus comparing said determining signature with the signature of the mixture produced and determined in an analogous manner from the allele whereby an identity of said signatures indicates the presence of the allele and nonidentity of the signatures indicates the absence of the allele.
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10. A method to determined the presence or absence of an infectious agent in a biological sample which method comprises:
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subjecting said sample to the polymerase chain reaction using primers characteristics of the suspected infectious agent and purifying the reaction product to obtain a purified DNA segment; subjecting said segment to replication in a process consisting essentially of multiple cycles of replication in the presence of a single primer fully complementary to a portion consisting of at least about ten nucleotides of said segment, wherein said portion is not a repetitive sequence or part of a gene cluster within said DNA segment, under conditions of low stringency at 25°
C.-40°
C., a primer concentration in the range of 2-10 μ
M, and a polymerase concentration in the range of 60-200 units/ml to obtain a mixture of replicated DNA molecules which result from said multiplicity of low stringency cycles of replication; anddetermining the sizes of the replicated DNA molecules in the mixture thus obtaining a signature characteristic of said segment comparing said determining signature with the signature of the mixture produced and determined in an analogous manner from the suspected infectious agent; wherein identity of the signature indicates the presence of said infectious agent.
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11. A method to determine the relationship of a subject through the maternal line to another individual which method comprises:
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obtaining mitochondrial DNA from said subject and from said other individual; obtaining in purified and cloned or amplified form DNA segments representing a defined region of the mitochondrial DNA of the subject and of said individual;
followed by a process consisting essentially ofsubjecting each segment to multiple cycles of replication in the presence of a single primer fully complementary to a portion consisting of at least about ten nucleotides of each said segment, wherein said portion is not a repetitive sequence or part of a gene cluster within said DNA segment, under conditions of low stringency at 25°
C.-40°
C., a primer concentration in the range of 2-10 μ
M, and a polymerase concentration in the range of 60-200 units/ml to obtain a mixture of replicated DNA molecules which result from said multiplicity of low stringency cycles of replication; anddetermining the sizes of the replicated DNA molecules in each said mixture thus obtaining a signature characteristic of each segment, and comparing the signature of the mixture obtained from the subject with the signature of the mixture obtained from the other individual; whereby identity of said signatures indicates relationship of the subject with the other individual through the maternal line.
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Specification