Process for amplifying, detecting, and/or cloning nucleic acid sequences

Claim 2

A process of Claim 1, wherein a sequence to be amplified and detected is contained within a larger sequence.

Claim 3

A process of Claim 1 or Claim 2, wherein the agent for polymerization is E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, reverse transcriptase, or a heat-stable enzyme.

Claim 4

A process of any one of Claims 1-3, wherein the nucleic acid is DNA and the primers are oligodeoxyribonucleotides.

Claim 5

A process of any one of Claims 1-4, wherein each primer employed contains a restriction site which is the same as or different from a restriction site the other primer, and after step (c) or (d) and before step (e) the product of step (c) or (d) (as the case may be) is cleaved with a restriction enzyme specific for each of said restriction sites and the cleaved products are separated from the uncleaved products and used in step (e).

Claim 6

A process of any one of Claims 1-5, wherein a sequence to be amplified and detected is associated with a genetic, cancerous or infectious disease.

Claim 7

A process of any one of Claims 1-6, wherein a single-stranded nucleic acid sequence which it is desired to detect is first treated under hybridizing conditions with a primer, inducing agent for polymerization and the different nucleoside triphosphates, to form a complementary strand thereto thereby to provide said starting double-stranded nucleic acid sequence and to permit detection of said single-stranded sequence.

Claim 8

A process for cloning into a vector a specific double-stranded nucleic acid sequence or sequences contained in a nucleic acid or a mixture of nucleic acids, which process comprises first amplifying said sequence or sequences, by the following steps: (a) separating the nucleic acid strands and treating the nucleic acid(s) with a molar excess of a pair of oligonucleotide primers for each different specific sequence being amplified and cloned, one for each strand, under hybridizing conditions and in the presence of an inducing agent for polymerization and the different nucleoside triphosphates such that for each of said strands an extension product of each primer is synthesized which is complementary to its respective strand, wherein said primers are selected so that each is substantially complementary to one end of the sequence to be amplified on one of the strands such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer of the pair and wherein said primers each contain a restriction site which is the same as or different from the restriction site(s) on the other primer(s); (b) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules; (c) treating as in (a) the single-stranded molecules generated from step (b) with oligonucleotide primers such that a primer extension product is synthesized using each of the single strands produced in step (b) as a template; and, if desired, (d) repeating steps (b) and (c) at least once; whereby exponential amplification of the nucleic acid sequence or sequences results; (e) adding to the product of step (c) or (d) a restriction enzyme for each of said restriction sites to obtain cleaved products in a restriction digest; and (f) ligating the cleaved product(s) into one or more cloning vectors.

Claim 9

A process for synthesizing a nucleic acid fragment from an existing double-stranded nucleic acid sequence having fewer nucleotides than the fragment being synthesized and two oligonucleotide primers, wherein the nucleic acid being synthesized is comprised of a left segment, a core segment and a right segment, and wherein the core segment represents at least substantially the nucleotide sequence of said existing nucleic acid sequence, and the right and left segments represent the nucleotide sequence present at one end of each of the two primers, the other ends of which are complementary or substantially complementary to ends of the single strands produced by separating the strands of said existing nucleic acid sequence, which process comprises: (a) treating the strands of said existing fragment with a molar excess of two oligonucleotide primers, one for each of the strands, under hybridizing conditions and in the presence of an inducing agent for polymerization and the different nucleoside triphosphates such that for each strand an extension product of the respective primer is synthesized which is complementary to the strand, wherein said primers are selected so as each to be substantially complementary to one end of a strand of said existing sequence such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of an extension product of the other primer, and wherein each primer also contains a sequence of nucleotides which are not complementary to said existing sequence and which correspond to the two ends of the nucleic acid fragment being synthesized; (b) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules; and (c) treating the single-stranded molecules generated from step (b) with the primers of step (a) under conditions such that a primer extension product is synthesized using each of the single strands produced in step (b) as a template so as to produce two intermediate double-stranded nucleic acid molecules, into each of which has been incorporated the nucleotide sequence present at one end of the oligonucleotide primers, and two full-length double-stranded nucleic acid molecules, into each of which has been incorporated the nucleotide sequence present at one end of both of the oligonucleotide primers; (d) repeating steps (b) and (c) for a sufficient number of times to produce the full-length double-stranded molecules in an effective amount.

Claim 10

A process as claimed in Claim 9, wherein the product of step (d) is then used to produce a yet further extended nucleic acid sequence by extending it in accordance with a process as defined in Claim 9.

Claim 11

A process as claimed in Claim 9 or Claim 10, wherein the existing nucleic acid sequence is obtained by treating two oligonucleotides, each of which has at one end thereof a nucleotide sequence which is complementary to an end of the other oligonucleotide and which are non-complementary to each other at their other ends, with an agent for polymerization and the different nucleoside triphosphates under conditions such that an extension product of each oligonucleotide is synthesized which is complementary to the other of the resulting extension products.

Claim 12

A process according to any one of Claims 8 to 10, wherein a single-stranded nucleic acid sequence is first treated under hybridizing conditions with a primer, inducing agent for polymerization and the different nucleoside triphosphate, to form a complementary strand thereto thereby to provide said starting double-stranded nucleic acid sequence.

Claim 13

A kit for the detection of at least one specific nucleic acid sequence in a sample, which kit comprises, in packaged form, a multicontainer unit having: (a) each of two oligonucleotide primers for each different sequence to be detected, wherein (i) if the specific nucleic acid sequence to be detected is single-stranded one primer is substantially complementary to one end of the strand so that an extension product of said one primer formed under hybridizing conditions and in the presence of an inducing agent for polymerization and the different nucleoside triphosphates is substantially complementary to said strand, and the other primer is substantially complementary to one end of said extension product and can be used under hybridizing conditions and in the presence of an inducing agent for polymerization and the different nucleoside triphosphates to synthesize another extension product employing said extension product of said one primer as a template thereby providing a nucleic acid consisting of two strands; or (ii) if the specific nucleic acid sequence to be detected is double-stranded the primers are such that each is substantially complementary to one end of one of the strands and an extension product synthesized from each primer using its complement strand as a template under hybridizing conditions and in the presence of an inducing agent for polymerization and the different nucleoside triphosphates, when separated from its complement, can serve as a template for synthesis of an extension product of the other primer under hybridizing conditions and in the presence of an inducing agent for polymerization and the different nucleoside triphosphates; (b) an agent for polymerization; (c) each of the different nucleoside triphosphates; (d) an oligonucleotide probe capable of hybridizing to said sequence if it is present in said sample; and (e) means for detecting hybrids of said probe and said sequence.