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

Claim 2

A process for detecting the presence or absence of at least one specific nucleic acid sequence in a sample containing a nucleic acid or mixture of nucleic acids, or distinguishing between two different sequences in said sample, wherein the sample is suspected of containing said sequence or sequences, which process comprises: (a) treating the sample with one oligonucleotide primer for each strand of each different specific sequence being amplified, under hybridizing conditions such that for each strand of each different sequence being amplified an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primer or primers are selected so as to be substantially complementary to each strand of each specific 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 the extension product of the other primer; (b) treating the sample under denaturing conditions to separate the primer extension products from their templates if the sequence or sequences to be detected are present; (c) treating the sample with oligonucleotide primers such that a primer extension product is synthesized using each of the single strands produced in step (b) as a template, resulting in amplification of the specific nucleic acid sequence or sequences if present; (d) adding to the product of step (c) a labeled oligonucleotide probe for each sequence being detected capable of hybridizing to said sequence or a mutation thereof; and (e) determining whether said hybridization has occurred.

Claim 3

The process of claim 1, wherein steps (b) and (c) are repeated at least once and steps (a) and - (c) are accomplished by treatment with four different nucleotide triphosphates and an agent for polymerization, which are added together with or separately from the primer(s).

Claim 4

The process of claim 2, wherein the agent for polymerization is E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase or reverse transcriptase.

Claim 5

The process of any one of claims 1-3, wherein the nucleic acid is double stranded and its strands are separated by denaturing before of during step (a).

Claim 6

The process of any one of claims 1-4, wherein the nucleic acid is DNA and the primers are oligodeoxyribonucleotides.

Claim 7

The process of any one of claims 1-5, wherein each primer employed contains a restriction site on its 5' end which is the same as or different from a restriction site on another primer, and after step (c) and before step (d) the product of step (c) is cleaved with a restriction enzyme specific for each of said restriction sites and the cleaved products are separated from the un- cleaved products and used in step (d).

Claim 8

The process of any one of claims 1-6, wherein the specific nucleic acid sequence is associated with a genetic, cancerous or infectious disease.

Claim 9

A kit for the detection of at least one specific nucleic acid sequence in a sample containing one or more nucleic acids at least one of which nucleic acid is supected of containing said sequence, which kit comprises, in packaged form, a multicontainer unit having: (a) one container for each oligonucleotide primer for each strand of each different sequence to be detected, which primer or primers are substantially complementary to each strand of each specific nucleic acid sequence such that an extension product synthesized from one primer, when it is separated from its complement, can serve as a template for the synthesis of the extension product of the-other primer; (b) a container containing an agent for polymerization; (c) a container for each of four different nucleoside triphosphates; (d) a container containing a labeled oligonucleotide probe capable of hybridizing to said sequence if it is present in said sample; and (e) a container containing means for detecting hybrids of said probe and said sequence.

Claim 10

A kit for the detection of at least one specific nucleic acid sequence in a sample containing one or more nucleic acids at least one of which nucleic acid is supected of containing said sequence, which kit comprises, in packaged form, a multicontainer unit having: (a) one container for each oligonucleotide primer for each strand of each different sequence to be detected, which primer or primers are substantially complementary to each strand of each specific nucleic acid sequence such that an extension product synthesized from one primer, when it is separated from its complement, can serve as a template for the synthesis of the extension product of the-other primer; (b) a container containing an agent for polymerization; (c) a container for each of four different nucleoside triphosphates; (d) a container containing a labeled oligonucleotide probe capable of hybridizing to said sequence if it is present in said sample; and (e) a container containing means for detecting hybrids of said probe and said sequence.

Claim 11

A process for cloning into a vector a specific nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids, which process comprises: (a) treating the nucleic acid(s) with one oligonucleotide primer for each strand of each different specific sequence being amplified, under conditions such that for each strand of each different sequence being amplified an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primer or primers are selected so as to be substantially complementary to each strand of each specific 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 the extension product of the other primer, and wherein said primer or primers each contain a restriction site on its 5' end 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 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, wherein depending on the particular sequence being amplified, steps (a) and (c) are carried out in the presence of from 0 up to an effective amount of dimethylsulfoxide or at a temperature of up to about 45°C. (d) adding to the product of step (c) a restriction enzyme for each of said restriction sites to obtain cleaved products in a restriction digest; and (e) ligating the cleaved product(s) into one or more cloning vectors.

Claim 12

A process for cloning into a vector a specific nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids, which process comprises: (a) treating the nucleic acid(s) with one oligonucleotide primer for each strand of each different specific sequence being amplified, under conditions such that for each strand of each different sequence being amplified an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primer or primers are selected so as to be substantially complementary to each strand of each specific 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 the extension product of the other primer, and wherein said primer or primers each contain a restriction site on its 5' end 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 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, wherein depending on the particular sequence being amplified, steps (a) and (c) are carried out in the presence of from 0 up to an effective amount of dimethylsulfoxide or at a temperature of up to about 45°C. (d) adding to the product of step (c) a restriction enzyme for each of said restriction sites to obtain cleaved products in a restriction digest; and (e) ligating the cleaved product(s) into one or more cloning vectors.

Claim 13

A process for synthesizing a nucleic acid fragment from an existing nucleic acid fragment 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 fragment, and the right and left segments represent the nucleotide sequence present in the 5' ends of the two primers, the 3' ends of which are complementary or substantially complementary to the 3' ends of the single strands produced by separating the strands of said existing nucleic acid fragment, which process comprises: (a) treating the strands of said existing fragment with two oligonucleotide primers under conditions such that an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primers are selected so as to be substantially complementary to the 3' end of each strand of said existing fragment 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, and wherein each primer contains, at its 5' end, a sequence of nucleotides which are not complementary to said existing fragment 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 in the 5' end of one of the oligonucleotide primers, and two full-length double-stranded nucleic acid molecules, into each of which has been incorporated the nucleotide sequence present in the 5' ends 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;; (e) treating the strands of the product of step (d) with two primers so as to lengthen the product of step (d) on both ends; and (f) repeating steps (a)-(d) using the product of step (d) as the core fragment and two oligonucleotide primers which are complementary. or substantially complementary to the 3' ends of the single strands produced by separating the strands of the product of step (d).

Claim 14

A process for synthesizing a nucleic acid fragment from an existing nucleic acid fragment 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 fragment, and the right and left segments represent the nucleotide sequence present in the 5' ends of the two primers, the 3' ends of which are complementary or substantially complementary to the 3' ends of the single strands produced by separating the strands of said existing nucleic acid fragment, which process comprises: (a) treating the strands of said existing fragment with two oligonucleotide primers under conditions such that an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primers are selected so as to be substantially complementary to the 3' end of each strand of said existing fragment 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, and wherein each primer contains, at its 5' end, a sequence of nucleotides which are not complementary to said existing fragment 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 in the 5' end of one of the oligonucleotide primers, and two full-length double-stranded nucleic acid molecules, into each of which has been incorporated the nucleotide sequence present in the 5' ends 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;; (e) treating the strands of the product of step (d) with two primers so as to lengthen the product of step (d) on both ends; and (f) repeating steps (a)-(d) using the product of step (d) as the core fragment and two oligonucleotide primers which are complementary. or substantially complementary to the 3' ends of the single strands produced by separating the strands of the product of step (d).