Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme

A process for amplifying at least one specific DNA sequenc contained in a DNA or a mixture of nucleic acids, wherein the DNA consists of two complementary strands of equal or unequal length, which process comprises:

• (a) heating the DNA in the presence of four different nucleoside triphosphates and two oligonucleotide primers, for each different specific sequencing being amplified, for an effective time and at an effective temperature to denature each nucleic acid, wherein each primer is selected to be sufficiently complementary to different strands of each specific sequence to hybridize therewith, such that the extension product synthesized from one primer, when separated from its complement, can serve as a template for synthesis of the extension product of the other primer;
• (b) cooling the denatured DNA to temperature which promotes hybridization of each primer to its complementary strand;
• (c) contacting the denatured DNA, at the same time as or after step (a) or (b), with a thermostable enzyme which enables combination of the nucleoside triphosphates to form primer extension products complementary to each strand of DNA;
• (d) maintaining the mixture from step (c) at an effective temperature for an effective time to promote the activity of the thermostable enzyme, and to synthesize, for each different sequence being amplified, an extension product of each primer which si complementary to each strand, but not so high as to separate each extension product from its complementary strand;
• (e) heating the mixture from step (d) for an effective time and at an effective temperature to separate the primer extension products from the strands on which they were synthesized to produce single-stranded molecules, but not so high as to denature irreversibly the enzyme;
• (f) cooling the mixture from step (e) for an effective time and to an effective temperature to promote hybridization of the primer to its complementary single-stranded molecule produced in step (e);
• and
• (g) maintaining the mixture from step (f) at an effective temperature for an effective time to promote the activity of the enzyme, and to synthesize, for each different sequence being amplified, an extension product of each primer which is complementary to each strand produced in step (f), but not so high as to separate each extension product form its complementary strand, wherein steps (f) and (g) are carried out simultaneously or sequentially.

A process for detecting the presence or absence of at least one specific DNA sequence in a sample containing a DNA or mixture of nucleic acids, or distinguishing between two different DNA sequences in said sample, wherein the sample is suspected of containing said sequence or sequences, and wherein if the nucleic acid(s) are double-stranded, they each consist of two separated complementary strands of equal or unequal length, which process comprises:

• (a) contacting the sample with four different nucleoside triphosphates and two oligonucleotide primers, for each different specific sequence being detected, wherein each primer is selected to be sufficiently complementary to different strands of each specific sequence to hybridize therewith, such that the extension product synthesized from one primer, when separated from its complement, can serve as a template for synthesis of the extension product of the other primer, at a temperature which promotes hybridization of each primer to its complementary strand;
• (b) contacting the sample, at the same time as or after step (a), with a thermostable enzyme which catalyzes combination of the nucleoside triphosphates to form primer extension products complementary to each strand of DNA;
• (c) maintaining the mixture from step (b) at an effective temperature for an effective time to promote the activity of the enzyme, and to synthesize, for each different sequence being detected, an extension product of each primer which is complementary to each strand, but not so high as to separate each extension product from its complementary strand;
• (d) heating the mixture from step (c) for an effective time and at an effective temperature to separate the primer extension products from the strands on which they were synthesized to produce single-stranded molecules, but not so high as to denature irreversibly the thermostable enzyme;
• (e) cooling the mixture from step (d) for an effective time and to an effective temperature to promote hybridization of each primer to its complementary single-stranded molecule produced in step (d);
• (f) maintaining the mixture from step (e) at an effective temperature for an effective time to promote the activity of the enzyme and to synthesize, for each different sequence being detected, but not so high as to separate each extension product from its complementary strand, resulting in amplification in quantity of the specific nucleic acid sequence or sequences if present, wherein steps (e) and (f) are carried out simultaneously or sequentially;
• (g) adding to the product of step (f) a labeled oligonucleotide probe for each sequence being detected capable of hybridizing to said sequence or to a mutation thereof;
• and
• (h) determining whether said hybridization has occurred.

A process for detecting the presence or absence of at least one specific DNA sequence in a sample containing DNA or mixture of nucleic acids, or distinguishing between two different DNA sequences in said sample, where the sample is suspected of containing said sequence or sequences and the DNA is double-stranded, which process comprises:

• (a) heating the sample in the presence of four different nucleoside triphosphates and two oligonucleotide primers, for each different specific sequence being detected, for an effective time and at an effective temperature to denature the DNA in the sample, wherein each primer is selected to be sufficiently complementary to different strands of each specific sequence to hybridize therewith, such that the extension product synthesized from one primer, when separated from its complement, can serve as a template for synthesis of the extension product of the other primer,
• (b) cooling the denatured DNA to a temperature promotes hybridization of each primer to its complementary strand;
• (c) contacting the natured DNA, at the same time as or after step (a) or (b), with a thermostable enzyme which catalyzes combination of the nucleoside triphosphates to form primer extension products complementary to each strand of DNA;
• (d) maintaining the mixture from step (c) at an effective temperature for an effective time to promote the activity of the enzyme and to synthesize, for each different sequence being detected, an extension product of each primer which is complementary to each strand, but not so high as to separate each extension product from its complementary strand;
• (e) heating the mixture from step (d) for an effective time and at an effective temperature to separate the primer extension products from the strands on which they were synthesized to produce single-stranded molecules, but not so high as to denature irreversibly the enzyme;
• (f) cooling the mixture from step (e) for an effective time and to an effective temperature to promote hybridization of each primer to its complementary single-stranded molecule produced from step (e);
• (g) maintaining the mixture from step (f) at an effective temperature for an effective time to promote the activity of the enzyme and to synthesize, for each different sequence being detected, an extension product of each primer which is complementary to each strand, but not so high as to separate each extension product from its complementary strand, resulting in amplification in quantity of the specific nucleic acid sequence or sequences if present, wherein steps (f) and (g) are carried out simultaneously or sequentially;
• (h) adding to the product of step (g) a labeled oligonucleotide probe for each sequence being detected capable of hybridizing to said sequence or to a mutuation thereof;
• and
• (i) determining whether said hybridizing has occurred. determining whether said hybridization has occurred.

A process for cloning into a cloning vector one or more specific DNA sequences contained in a DNA or a mixture of nucleic acids, which DNA when double-stranded consists of two separated complementary strands, and which DNA is amplified in quantity before cloning, which process comprises:

• (a) contacting each DNA with four different nucleoside triphosphates and two oligonucleotide primers, for each different specific sequence being amplified, wherein each primer is selected to be sufficiently complementary to different strands of each specific sequence to hybridize therewith, such that the extension product synthesized from one primer, when separated from its complement, can serve as a template for synthesis of the extension product of the other primer, and wherein each sequence being amplified or each primer contains a restriction site, at a temperature which promotes hybridization of each primer to its complementary strand;
• (b) contacting each strand, at the same time as or after step (a) or (b), with a thermostable enzyme which catalyzes combination of the nucleoside triphosphates to form primer extension products complementary to each strand of DNA;
• (c) maintaining the mixture from step (b) at an effective temperature for an effective time to promote the activity of the enzyme and to synthesize, for each different sequence being amplified, an extension product of each primer which is complementary to each strand, but not so high as to separate each extension product from its complementary strand;
• (d) heating the mixture from step (c) for an effective time and at an effective temperature to separate the primer extension products from the strands on which they were synthesized, to produce single-stranded molecules, but not so high as to denature irreversibly the enzyme;
• (e) cooling the mixture from step (d) for an effective time and to an effective temperature to promote hybridization of each primer to its complementary single-stranded molecule produced in step (d);
• (f) maintaining the mixture from step (e) at an effective temperature for an effective time to promote the activity of the enzyme and to synthesize, for each different sequence being amplified, an extension product of each primer which is complementary to each strand produced in step (d), but not so high as to separate each extension product from its complementary strand, steps (d), (e) and (f) being repeated a sufficient number of times to result in detectable amplification of the DNA containing the sequence(s), wherein steps (e) and (f) are carried simultaneously or sequentially;
• (g) adding to the product of step (f) a restriction enzyme for each of said restriction sites to obtain cleaved products in a restriction digest;
• and
• (h) ligating the cleaved product(s) of step (g) containing the specific sequence(s) to be cloned into one or more cloning vectors.

A process for cloning into a vector at least one specific DNA sequence contained in a DNA or a mixture of nucleic acids, wherein the DNA consists of two complementary strands of equal or unequal length, and wherein the DNA is amplified in quantity before cloning, which process comprises:

• (a) heating the DNA in the presence of four different nucleoside triphosphates and two oligonucleotide primers, for each different specific sequence being amplified, for an effective time and at an effective temperature to denature the DNA, wherein each primer is selected to be sufficiently complementary to different strands of each specific sequence to hybridize therewith, such that the extension product synthesized from one primer, when separated from its complement, can serve as a template for synthesis of the extension product of the other primer, and wherein each sequence being amplified or each primer contains a restriction site;
• cooling the denatured DNA to a temperature effective to promote hybridization between each primer and its complementary strand;
• (c) contacting the denatured DNA, at the same time as or after step (a) or (b), with a thermostable enzyme which catalyzes combination of the nucleoside triphosphates to form primer extension products complementary to each strand of DNA;
• (d) maintaining the mixture from step (c) at an effective temperature for an effective time to promote the activity of the enzyme and to synthesize, for each different sequence being amplified, an extension product of each primer which is complementary to each strand, but not so high as to separate each extension product from its complementary strand;
• (e) heating the mixture from step (d) for an effective time and at an effective temperature to separate the primer extension products from the strands on which they were synthesized, to produce single-stranded molecules, but not so high as to denature irreversibly the enzyme;
• (f) cooling the mixture from step (e) for an effective time and to an effective temperature to promote hybridization of each primer to its complementary single-stranded molecule produced in step (e);
• (g) maintaining the mixture from step (f) at an effective temperature for an effective time to promote the activity of the enzyme and to synthesize, for each different sequence being amplified, an extension product of each primer which is complementary to each strand produced in step (e), but not so high as to separate each extension product from its complementary strand, steps (e), (f), and (g) being repeated a sufficient number of times to result in detectable amplification of the DNA containing the sequences(s), wherein steps (f) and (g) are carried out simultaneously or sequentially;
• (h) adding to the product of step (g) a restriction enzyme for each of said restriction sites to obtain cleaved products in a restriction digest;
• and
• (i) ligating the cleaved product(s) of step (h) containing the specific sequence(s) to be cloned into one or more cloning vectors containing a selectable marker.

A process for cloning into a cloning vector one or more specific DNA sequences contained in a DNA or mixture of nucleic acids, which DNA when double-stranded consists of two separated complementary strands of equal to unequal length and which DNA is amplified in quantity before cloning which process comprises:

• (a) contacting each DNA with four different nucleoside triphosphates and two oligonucleotide primers, for each different specific sequence being amplified, wherein each primer is selected to sufficiently complementary to different strands of each specific sequence to hybridize therewith, such that the extension product synthesized from one primer, when separatd from its complement, can serev as a template for synthesis of teh extension product of teh other primer, at a temperature which promotes hybridization of each primer to its complementary strand;
• (b) contacting each strand, at the same time as or after step (a) or (b), with a thermostable enzyme which catalyzes combination of the nucleoside triphosphates to form primer extension products complementary to each strand of DNA;
• (c) maintaining the mixture from step (b) at an effective temperature for an effective time to promote the activity of the enzyme and to synthesize, for each different sequence being amplified, an extension product of each primer which is complementary to each strand, but not so high as to separate each extension product from its complementary strand;
• (d) heating the mixture from step (c) for an effective time and at an effective temperature to separate the primer extension products from the strands on which they were synthesized to produce single-stranded molecules, but not so high as to denature irreversibly the enzyme;
• (e) cooling the mixture from step (d) for an effective time and to an effective temperature to promote hybridization of each primer to its complementary single-stranded molecule produced in step (d);
• (f) maintaining the mixture from step (e) at an effective temperature for an effective time to promote the activity of the enzyme and to synthesize, for each different sequence being amplified, an extension product of each primer which is complementary to each strand produced in step (d), but not so high as to separate each extension product from its complementary strand, steps (d), (e) and (f) being repeated a sufficient number of times to result in effective amplification of the DNA containing the sequence(s) for blunt-end ligation into one or more cloning vectors, wherein steps (e) and (f) are conducted simultaneously or sequentially;
• and
• (g) ligating the amplified specific sequence(s) to be cloned obtained from step (f) into one or more of said cloning vectors in the presence of a ligase, said amplified sequence(s) and vector(s) being present in sufficient amounts to effect the ligation.

A process for cloning into a cloning vector at least one specific DNA sequence contained in a DNA or mixture of nucleic acids, which DNA consists of two complementary strands of equal to unequal length and which DNA is amplified in quantity before cloning, which process comprises:

• (a) heating the DNA in the presence of four different nucleoside triphophates and two oligonucleotide primers, for each different specific sequencing being amplified, for an effective time and at an effective temperature to denature the DNA, wherein each primer is selected be sufficiently complementary to different strands of each specific sequence to hybridize therewith, such that the extension product synthesized from one primer, when separated from its complement, can serve as a template for synthesis of the extension product of the other primer;
• (b) cooling the denatured DNA to a temperature effective to promote hybridization between each primer and its complementary strand;
• (c) contacting the denatured DNA, at the same time as or after step (a) or (b), with a thermostable enzyme which catalyzes combination of the nucleoside triphosphates to form primer extension products complementary to each strand of DNA;
• (d) maintaining the mixture from step (c) at an effective temperature for an effective time to promote the activity of the enzyme and to synthesize, for each different sequence being amplified, an extension product of each primer which is complementary to each strand, but not so high as to separate each extension product from its complementary strand;
• (e) heating the mixture from step (d) for an effective time and at an effective temperature to separate the primer extension products from the strands on which they were synthesized, to produce single-stranded molecules, but not so high as to denature irreversibly the enzyme;
• (f) cooling the mixture from step (e) for an effective time and to an effective temperature to promote hybridization of each primer to its complementary single-stranded molecule produced in step (e);
• (g) maintaining the mixture from step (f) at an effective temperature for an effective time to promote the activity of the enzyme and to synthesize, for each different sequence being amplified, an extension product of each primer which is complementary to each strand produced in step (e), but not so high as to separate each extension product from its complementary strand, steps (e), (f) and (g) being repeated a sufficient number of times to result in effective amplification of the DNA containing each sequence for bluntend ligation into one or more cloning vectors, wherein steps (f) and (g) are conducted simultaneously or sequentially;
• and
• (h) ligating the amplified specific sequence(s) to be cloned obtained from step (g) into one or more of said cloning vectors in the presence of a ligase, said amplified sequence(s) and vector(s) being present in sufficient to effect the ligation.