Arbitrarily primed polymerase chain reaction method for fingerprinting genomes

Claim 1

A method of generating a set of discrete DNA segments characteristic of a genome comprising: (a) forming a polymerase chain reaction (PCR) admixture by combining, in a PCR buffer, genomic DNA and at least one first polynucleotide primer from about 10 to about 50 nucleotide bases in length; (b) subjecting said PCR admixture of step (a) to at least one PCR thermocycle, each of said thermocycles comprising hybridization, primer extension and denaturation phases, said hybridization phase comprising hybridization conditions permitting the arbitrary priming of said genomic DNA, thereby producing said set of discrete DNA segments; (c) contacting, in a PCR buffer, the set of discrete DNA amplification products formed in step (b) with at least one second polynucleotide primer from about 10 to 50 nucleotides in length, wherein said second primer or primers of this step each have nucleotide sequences that match the first primer or primers used in step (a) except that the second primer(s) each have one or more additional nucleotide bases at the 3' terminus of each second primer, wherein the additional nucleotide bases are additional with respect to the 3' terminus of the first primer or primers, to form a second PCR admixture; and (d) subjecting said second PCR admixture to a plurality of PCR thermocycles, each of said thermocycles including hybridization, primer extension and denaturation phases, said hybridization phase comprising hybridization conditions that do not permit the formation of primer-template duplexes with a substantial degree of mismatching, thereby amplifying a subset of said discrete DNA segments.

Claim 2

A method of generating a set of discrete DNA segments characteristic of a genome comprising: (a) forming a polymerase chain reaction (PCR) admixture by combining, in a PCR buffer, genomic DNA and at least one first polynucleotide primer from about 10 to about 50 nucleotide bases in length; (b) subjecting said PCR admixture of step (a) to at least one PCR thermocycle, each of said thermocycles comprising hybridization, primer extension and denaturation phases, said hybridization phase comprising hybridization conditions permitting the arbitrary priming of said genomic DNA, thereby producing said set of discrete DNA segments; (c) contacting, in a PCR buffer, the set of discrete DNA amplification products formed in step (b) with at least one second polynucleotide primer from about 10 to 50 nucleotides in length, wherein said second primer or primers of this step each have nucleotide sequences that match the first primer or primers used in step (a) except that the second primer(s) each have one or more additional nucleotide bases at the 3' terminus of each second primer, wherein the additional nucleotide bases are additional with respect to the 3' terminus of the first primer or primers, to form a second PCR admixture; and (d) subjecting said second PCR admixture to a plurality of PCR thermocycles, each of said thermocycles including hybridization, primer extension and denaturation phases, said hybridization phase comprising hybridization conditions that do not permit the formation of primer-template duplexes with a substantial degree of mismatching, thereby amplifying a subset of said discrete DNA segments.

Claim 12

A method for generating a discrete set of DNA segments characteristic of a sample of single-stranded RNA, which method comprises: (a) forming a primer extension reaction admixture by combining, in a primer extension buffer, said RNA sample and at least one first polynucleotide primer from about 10 to about 50 nucleotide bases in length; (b) maintaining said primer extension reaction under primer extension conditions to produce a hybrid DNA-RNA molecule; (c) forming a polymerase chain reaction (PCR) admixture by combining, in a PCR buffer, said DNA-RNA hybrid molecule and at least one second polynucleotide primer from about 10 to about 50 nucleotide bases in length, wherein the first and second primer may be the same or different from one another; (d) subjecting said PCR admixture of step (c) to at least one PCR thermocycle, each of said thermocycles comprising hybridization, primer extension and denaturation phases, said hybridization phase comprising hybridization conditions compatible with arbitrary priming of said DNA-RNA hybrid molecule, thereby producing said set of discrete DNA segments; (e) contacting, in a PCR buffer, the set of discrete DNA amplification products formed in step (d) with at least one third polynucleotide primer from about 10 to 50 nucleotides in length, wherein said third primer or primers of this step each have nucleotide sequences that match the primer or primers used in step (c) except that the third primer(s) each have one or more additional nucleotide bases at the 3' terminus of each primer, wherein the additional nucleotide bases are additional with respect to the 3' terminus of the second primer or primers, to form a second PCR admixture; and (f) subjecting said second PCR admixture to a plurality of PCR thermocycles, each of said thermocycles including hybridization, primer extension and denaturation phases, said hybridization phase comprising hybridization conditions that do not permit the formation of primer-template duplexes with a substantial degree of mismatch, thereby amplifying a subset of said discrete DNA segments.

Claim 13

A method for generating a discrete set of DNA segments characteristic of a sample of single-stranded RNA, which method comprises: (a) forming a primer extension reaction admixture by combining, in a primer extension buffer, said RNA sample and at least one first polynucleotide primer from about 10 to about 50 nucleotide bases in length; (b) maintaining said primer extension reaction under primer extension conditions to produce a hybrid DNA-RNA molecule; (c) forming a polymerase chain reaction (PCR) admixture by combining, in a PCR buffer, said DNA-RNA hybrid molecule and at least one second polynucleotide primer from about 10 to about 50 nucleotide bases in length, wherein the first and second primer may be the same or different from one another; (d) subjecting said PCR admixture of step (c) to at least one PCR thermocycle, each of said thermocycles comprising hybridization, primer extension and denaturation phases, said hybridization phase comprising hybridization conditions compatible with arbitrary priming of said DNA-RNA hybrid molecule, thereby producing said set of discrete DNA segments; (e) contacting, in a PCR buffer, the set of discrete DNA amplification products formed in step (d) with at least one third polynucleotide primer from about 10 to 50 nucleotides in length, wherein said third primer or primers of this step each have nucleotide sequences that match the primer or primers used in step (c) except that the third primer(s) each have one or more additional nucleotide bases at the 3' terminus of each primer, wherein the additional nucleotide bases are additional with respect to the 3' terminus of the second primer or primers, to form a second PCR admixture; and (f) subjecting said second PCR admixture to a plurality of PCR thermocycles, each of said thermocycles including hybridization, primer extension and denaturation phases, said hybridization phase comprising hybridization conditions that do not permit the formation of primer-template duplexes with a substantial degree of mismatch, thereby amplifying a subset of said discrete DNA segments.

Claim 15

A method for generating a discrete set of DNA segments characteristic of a sample of single-stranded RNA, which method comprises: (a) forming a polymerase chain reaction (PCR) admixture by combining said RNA sample, at least one polynucleotide primer from about 10 to about 50 nucleotide bases in length, and a PCR buffer that contains both reverse transcriptase and thermostable DNA polymerase; (b) maintaining said PCR admixture under reverse transcriptase primer extension conditions to produce a hybrid DNA-RNA molecule; (c) subjecting said PCR admixture of step (b) to at least one PCR thermocycle, each of said thermocycles comprising hybridization, primer extension and denaturation phases, said hybridization phase comprising hybridization conditions compatible with arbitrary priming of said DNA-RNA hybrid molecule, thereby producing said set of discrete DNA segments; (d) contacting, in a PCR buffer, the set of discrete DNA amplification products formed in step (c) with at least one polynucleotide primer from about 10 to 50 nucleotides in length, wherein said primer or primers of this step each have nucleotide sequences that match the primer or primers used in step (a) except that the primer(s) step each have one or more additional nucleotide bases at the 3' terminus of each primer to form a second PCR admixture; and (e) subjecting said second PCR admixture to plurality of PCR thermocycles, each of said thermocycles including hybridization, primer extension and denaturation phases, said hybridization phase comprising hybridization conditions that do not permit the formation of primer-template duplexes with a substantial degree of mismatch, thereby amplifying a subset of said discrete DNA segments.

Claim 16

A method for generating a discrete set of DNA segments characteristic of a sample of single-stranded RNA, which method comprises: (a) forming a polymerase chain reaction (PCR) admixture by combining said RNA sample, at least one polynucleotide primer from about 10 to about 50 nucleotide bases in length, and a PCR buffer that contains both reverse transcriptase and thermostable DNA polymerase; (b) maintaining said PCR admixture under reverse transcriptase primer extension conditions to produce a hybrid DNA-RNA molecule; (c) subjecting said PCR admixture of step (b) to at least one PCR thermocycle, each of said thermocycles comprising hybridization, primer extension and denaturation phases, said hybridization phase comprising hybridization conditions compatible with arbitrary priming of said DNA-RNA hybrid molecule, thereby producing said set of discrete DNA segments; (d) contacting, in a PCR buffer, the set of discrete DNA amplification products formed in step (c) with at least one polynucleotide primer from about 10 to 50 nucleotides in length, wherein said primer or primers of this step each have nucleotide sequences that match the primer or primers used in step (a) except that the primer(s) step each have one or more additional nucleotide bases at the 3' terminus of each primer to form a second PCR admixture; and (e) subjecting said second PCR admixture to plurality of PCR thermocycles, each of said thermocycles including hybridization, primer extension and denaturation phases, said hybridization phase comprising hybridization conditions that do not permit the formation of primer-template duplexes with a substantial degree of mismatch, thereby amplifying a subset of said discrete DNA segments.