US 4,683,195 A · Process for amplifying, detecting, and/or-cloning nucleic acid sequences

Abstract

The present invention is directed to a process for amplifying and detecting any target nucleic acid sequence contained in a nucleic acid or mixture thereof. The process comprises treating separate complementary strands of the nucleic acid with a molar excess of two oligonucleotide primers, extending the primers to form complementary primer extension products which act as templates for synthesizing the desired nucleic acid sequence, and detecting the sequence so amplified. The steps of the reaction may be carried out stepwise or simultaneously and can be repeated as often as desired. In addition, a specific nucleic acid sequence may be cloned into a vector by using primers to amplify the sequence, which contain restriction sites on their non-complementary ends, and a nucleic acid fragment may be prepared from an existing shorter fragment using the amplification process.

Claims26

Dependent claims

A process for detecting the presence or absence of at least one specific nuclei 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, under hybridizing conditions such that for each strand of each different sequence to which an oligonucleotide primer is hybridized 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 sufficiently complementary to each strand of each specific sequence to hybridize therewith 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 olignonucleotide 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.

3 more elements

A process for detecting the presence or absence of a nucleic acid sequence containing a polymorphic restriction site specific for sickle cell anemia which sequence is suspected of being contained in a sample, which process comprises:

(a) treating the sample, together or separately, with an oligodeoxyribonucleotide primer for each strand, four different nucleoside triphosphates, and an agent for polymerization under hybridizing conditions, such that for each strand of the nucleic acid sequence an extension product of each primer is synthesized which is sufficiently complementary to each strand of the nucleic acid sequence being detected to hybridize therewith and contains the region of the .beta.-globin gene known potentially to contain the mutation that causes sickle cell anemia, wherein said primers are selected 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 the templates on which they are synthesized if the sequence to be detected is present;
(c) treating the product of step (b) with oligodeoxyribonucleotide primers, four different nucleoside triphosphates, and an agent polymerization 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 sequence to be detected if present;
(d) hybridizing said primer extension products of step (c) with a labeled oligodeoxyribonucleotide probe complementary to a normal .beta.-globin gene;
(e) digesting the hybridized mixture from step (d) with a restriction enzyme for the restriction site specific for sickle cell anemia;
and
(f) detecting whether the digest contains a restriction fragment correlated with the presence of sickle cell anemia.

4 more elements

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 is sufficiently complementary to the nucleotide sequence of said existing nucleic acid fragment to hybridize therewith, 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 to, or sufficiently complementary to hybridize with, 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 sufficiently complementary to the 3' end of each strand of said existing fragment to hybridize therewith, 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;
(c) treating the single-stranded molecules generared 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 molecule 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 to, or sufficiently complementary to hybridize with, the 3' ends of the single strands produced by separating the strands of the product of step (d).

4 more elements

Dependencies are inferred from the claim text.

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

Life of this patent

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

Life of this patent

Abstract

Claims26