Purified thermostable enzyme and process for amplifying, detecting, and/or cloning nucleic acid sequences using said enzyme

Claim 2

A purified thermostable enzyme that catalyzes combination of nucleotide triphosphates to form a nucleic acid strand complementary to a nucleic acid template strand.

Claim 3

An enzyme according to claim 1 that is DNA polymerase.

Claim 4

An enzyme according to claim 1 or 2 that has a molecular weight of about 86,000 to 90,000 daltons.

Claim 5

An enzyme according to claim 3 from Thermus aquaticus .

Claim 6

An enzyme according to any one of claims 1-4 having at least 50% of the activity at pH 6.4 that it has at pH 8.0.

Claim 7

An enzyme according to any one of claims 1-5 that is in native form.

Claim 8

A gene encoding the enzyme of claim 2.

Claim 9

A gene according to claim 7 that was cloned from the genome of Thermus aquaticus .

Claim 10

A gene according to claim 8 that encodes an enzyme having a molecular weight of about 86,000-90,000 daltons.

Claim 11

A gene according to claim 8 that encodes an enzyme having a molecular weight of about 60,000-65,000 daltons.

Claim 12

A gene according to claim 9 that is contained within an approximately 3.5 kb Bgl II- Asp 718 (partial) restriction fragment of either Thermus aquaticus or bacteriophage CH35:Taq#4-2.

Claim 13

A gene according to claim 10 that is contained within an approximately 2.8 kb Hin dIII- Asp 718 restriction fragment of either the genome of Thermus aquaticus or the plasmid pFC85.

Claim 14

A plasmid selected from pFC85 or pFC83.(obtainable from ATCC 67422 or ATCC 67421, respectively),

Claim 15

A plasmid selected from pFC85 or pFC83.(obtainable from ATCC 67422 or ATCC 67421, respectively),

Claim 16

Bacteriophage CH35:Taq#4-2.(obtainable from ATCC 40336).

Claim 17

Bacteriophage CH35:Taq#4-2.(obtainable from ATCC 40336).

Claim 18

A stable enzyme composition comprising an enzyme according to any one of claims 1-6 in a buffer comprising one or more non-ionic polymeric detergents.

Claim 19

A composition according to claim 15 wherein the detergents are each present in a concentration of about 0.1% to about 0.5% volume/volume of the total composition.

Claim 20

A composition according to claim 15 or 16 wherein the detergents are a polyoxyethylated sorbitan monolaurate and an ethoxylated nonyl phenol.

Claim 21

A composition according to any one of claims 15-17 wherein the buffer comprises glycerol, Tris-Cl, pH 8.0, ethylenediamine tetraacetic acid, dithiothreitol, a polyoxyvethylated sorbitan monolaurate, an ethoxylated nonyl phenol, and gelatin.

Claim 22

A process for amplifying at least one specific nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids, wherein if the nucleic acid is double-stranded, it consists of two separated complementary strands of equal or unequal length, which process comprises: (a) contacting each nucleic acid strand with four different nucleotide triphosphates and one oligonucleotide primer for each different specific sequence being amplified, wherein each primer is selected to be substantially complementary to different strands 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, said contacting being at a temperature which promotes hybridization of each primer to its complementary nucleic acid strand; (b) contacting each nucleic acid strand, at the same time as or after step (a), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (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 nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (d) heating the mixture from step (c) for an effective time and at an effective temperature to separate the primer extension products from the templates 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) to an effective temperature to promote hybridization of each primer to each of the single-stranded molecules produced in step (d); and (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 nucleic acid strand template produced in step (d), but not so high as to separate each extension product from its complementary strand template, wherein steps (e) and (f) are conducted simultaneously or sequentially.

Claim 23

A process for amplifying at least one specific nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids, wherein if the nucleic acid is double-stranded, it consists of two separated complementary strands of equal or unequal length, which process comprises: (a) contacting each nucleic acid strand with four different nucleotide triphosphates and one oligonucleotide primer for each different specific sequence being amplified, wherein each primer is selected to be substantially complementary to different strands 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, said contacting being at a temperature which promotes hybridization of each primer to its complementary nucleic acid strand; (b) contacting each nucleic acid strand, at the same time as or after step (a), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (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 nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (d) heating the mixture from step (c) for an effective time and at an effective temperature to separate the primer extension products from the templates 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) to an effective temperature to promote hybridization of each primer to each of the single-stranded molecules produced in step (d); and (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 nucleic acid strand template produced in step (d), but not so high as to separate each extension product from its complementary strand template, wherein steps (e) and (f) are conducted simultaneously or sequentially.

Claim 24

A process for amplifying at least one specific nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids, wherein the nucleic acid consists of two complementary strands of equal or unequal length, which process comprises: (a) heating each nucleic acid in the presence of four different nucleotide triphosphates and one oligonucleotide primer for each different specific sequence being amplified, for an effective time and at an effective temperature to denature each nucleic acid, wherein each primer is selected to be substantially complementary to different strands 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) cooling the denatured nucleic acids to a temperature which promotes hybridization of each primer to its complementary nucleic acid strand; (c) contacting the denatured nucleic acids, at the same time as or after step (a) or (b), with a thermostable enzyme which enables combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (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 is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (e) heating the mixture from step (d) for an effective time and at an effective temperature to separate the primer extension products from the templates 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 nucleic acid strand template produced in step (f), but not so high as to separate each extension product from its complementary strand template, wherein steps (f) and (g) are carried out simultaneously or sequentially.

Claim 25

A process for amplifying at least one specific nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids, wherein the nucleic acid consists of two complementary strands of equal or unequal length, which process comprises: (a) heating each nucleic acid in the presence of four different nucleotide triphosphates and one oligonucleotide primer for each different specific sequence being amplified, for an effective time and at an effective temperature to denature each nucleic acid, wherein each primer is selected to be substantially complementary to different strands 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) cooling the denatured nucleic acids to a temperature which promotes hybridization of each primer to its complementary nucleic acid strand; (c) contacting the denatured nucleic acids, at the same time as or after step (a) or (b), with a thermostable enzyme which enables combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (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 is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (e) heating the mixture from step (d) for an effective time and at an effective temperature to separate the primer extension products from the templates 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 nucleic acid strand template produced in step (f), but not so high as to separate each extension product from its complementary strand template, wherein steps (f) and (g) are carried out simultaneously or sequentially.

Claim 26

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, 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 nucleotide triphosphates and one oligonucleotide primer, for each different specific sequence being detected, wherein each primer is selected to be substantially complementary to different strands 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, said contacting being at a temperature which promotes hybridization of each primer to its complementary nucleic acid strand; (b) contacting the sample, at the same time as or after step (a), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (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 nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (d) heating the mixture from step (c) for an effective time and at an effective temperature to separate the primer extension products from the templates 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, an extension product of each primer which is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template, 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.

Claim 27

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, 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 nucleotide triphosphates and one oligonucleotide primer, for each different specific sequence being detected, wherein each primer is selected to be substantially complementary to different strands 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, said contacting being at a temperature which promotes hybridization of each primer to its complementary nucleic acid strand; (b) contacting the sample, at the same time as or after step (a), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (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 nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (d) heating the mixture from step (c) for an effective time and at an effective temperature to separate the primer extension products from the templates 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, an extension product of each primer which is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template, 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.

Claim 28

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 and the nucleic acid(s) are double-stranded, which process comprises: (a) heating the sample in the presence of four different nucleotide triphosphates and one oligonucleotide primer, for each different specific sequence being detected, for an effective time and at an effective temperature to denature each nucleic acid in the sample, wherein each primer is selected to be substantially complementary to different strands 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) cooling the denatured nucleic acids to a temperature which promotes hybridization of each primer to its complementary nucleic acid sequence; (c) contacting the denatured nucleic acids, at the same time as or after step (a) or (b), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (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 nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (e) heating the mixture from step (d) for an effective time and at an effective temperature to separate the primer extension products from the templates 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 nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template, 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 (9) a labeled oligonucleotide probe for each sequence being detected capable of hybridizing to said sequence or to a mutation thereof; and (i) determining whether said hybridization has occurred.

Claim 29

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 and the nucleic acid(s) are double-stranded, which process comprises: (a) heating the sample in the presence of four different nucleotide triphosphates and one oligonucleotide primer, for each different specific sequence being detected, for an effective time and at an effective temperature to denature each nucleic acid in the sample, wherein each primer is selected to be substantially complementary to different strands 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) cooling the denatured nucleic acids to a temperature which promotes hybridization of each primer to its complementary nucleic acid sequence; (c) contacting the denatured nucleic acids, at the same time as or after step (a) or (b), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (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 nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (e) heating the mixture from step (d) for an effective time and at an effective temperature to separate the primer extension products from the templates 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 nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template, 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 (9) a labeled oligonucleotide probe for each sequence being detected capable of hybridizing to said sequence or to a mutation thereof; and (i) determining whether said hybridization has occurred.

Claim 30

A process for detecting the presence or absence of at least one nucleotide variation in sequence in one or more nucleic acids contained in a sample, wherein if the nucleic acid is double-­stranded, it consists of two separated complementary strands of equal or unequal length, which process comprises: (a) contacting the sample with four different nucleotide triphosphates and one oligonucleotide primer, for each strand of each nucleic acid suspected of containing said variation, wherein each primer is selected to be substantially complementary to different strands 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, said contacting being at a temperature which promotes hybridization of each primer to its complementary nucleic acid strand; (b) contacting the sample, at the same time as or after step (a), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (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 nucleic acid suspected of containing said variation(s), an extension product of each primer which is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (d) heating the mixture from step (c) for an effective time and at an effective temperature to separate the primer extension products from the templates 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 nucleic acid suspected of containing said variation(s), an extension product of each primer which is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template, wherein steps (d), (e) and (f) are repeated a sufficient number of times to result in detectable amplification of the nucleic acid containing the sequence variation(s), if present, and wherein steps (e) and (f) are carried out simultaneously or sequentially; (g) affixing the product of step (f) to a membrane; (h) treating the membrane under hybridization conditions with a labeled sequence-specific oligonucleotide probe capable of hybridizing with the amplified nucleic acid sequence only if a sequence of the probe is complementary to a region of the amplified sequence; and; (i) detecting whether the probe has hybridized to an amplified sequence in the nucleic acid sample.

Claim 31

A process for detecting the presence or absence of at least one nucleotide variation in sequence in one or more nucleic acids contained in a sample, wherein if the nucleic acid is double-­stranded, it consists of two separated complementary strands of equal or unequal length, which process comprises: (a) contacting the sample with four different nucleotide triphosphates and one oligonucleotide primer, for each strand of each nucleic acid suspected of containing said variation, wherein each primer is selected to be substantially complementary to different strands 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, said contacting being at a temperature which promotes hybridization of each primer to its complementary nucleic acid strand; (b) contacting the sample, at the same time as or after step (a), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (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 nucleic acid suspected of containing said variation(s), an extension product of each primer which is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (d) heating the mixture from step (c) for an effective time and at an effective temperature to separate the primer extension products from the templates 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 nucleic acid suspected of containing said variation(s), an extension product of each primer which is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template, wherein steps (d), (e) and (f) are repeated a sufficient number of times to result in detectable amplification of the nucleic acid containing the sequence variation(s), if present, and wherein steps (e) and (f) are carried out simultaneously or sequentially; (g) affixing the product of step (f) to a membrane; (h) treating the membrane under hybridization conditions with a labeled sequence-specific oligonucleotide probe capable of hybridizing with the amplified nucleic acid sequence only if a sequence of the probe is complementary to a region of the amplified sequence; and; (i) detecting whether the probe has hybridized to an amplified sequence in the nucleic acid sample.

Claim 32

A process for detecting the presence or absence of at least one nucleotide variation in sequence in one or more nucleic acids contained in a sample, wherein the nucleic acid(s) each consist of two complementary strands of equal or unequal length, which process comprises: (a) heating the sample in the presence of four different nucleotide triphosphates and one oligonucleotide primer, for each strand of each nucleic acid suspected of containing said variation(s), for an effective time and at an effective temperature to denature each nucleic acid, wherein each primer is selected to be substantially complementary to different strands of each specific nucleic acid, 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) cooling the denatured nucleic acids to a temperature which promotes hybridization of each primer to its complementary nucleic acid strand; (c) contacting the denatured nucleic acids, at the same time as or after step (a) or (b), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (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 nucleic acid suspected of containing said variation(s), an extension product of each primer which is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; e) heating the mixture from step (d) for an effective time and at an effective temperature to separate the primer extension products from the templates 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 nucleic acid suspected of containing said variation(s), an extension product of each primer which is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template, steps (e)-(g) being repeated a sufficient number of times to result in detectable amplification of the nucleic acid containing the sequence variation(s), if present, wherein steps (f) and (g) are carried out simultaneously or sequentially; (h) affixing the product of step (g) to a membrane; (i) treating the membrane under hybridization conditions with a labeled sequence-specific oligonucleotide probe capable of hybridizing with the amplified nucleic acid sequence only if a sequence of the probe is complementary to a region of the amplified sequence; and (j) detecting whether the probe has hybridized to an amplified sequence in the nucleic acid sample.

Claim 33

A process for detecting the presence or absence of at least one nucleotide variation in sequence in one or more nucleic acids contained in a sample, wherein the nucleic acid(s) each consist of two complementary strands of equal or unequal length, which process comprises: (a) heating the sample in the presence of four different nucleotide triphosphates and one oligonucleotide primer, for each strand of each nucleic acid suspected of containing said variation(s), for an effective time and at an effective temperature to denature each nucleic acid, wherein each primer is selected to be substantially complementary to different strands of each specific nucleic acid, 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) cooling the denatured nucleic acids to a temperature which promotes hybridization of each primer to its complementary nucleic acid strand; (c) contacting the denatured nucleic acids, at the same time as or after step (a) or (b), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (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 nucleic acid suspected of containing said variation(s), an extension product of each primer which is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; e) heating the mixture from step (d) for an effective time and at an effective temperature to separate the primer extension products from the templates 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 nucleic acid suspected of containing said variation(s), an extension product of each primer which is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template, steps (e)-(g) being repeated a sufficient number of times to result in detectable amplification of the nucleic acid containing the sequence variation(s), if present, wherein steps (f) and (g) are carried out simultaneously or sequentially; (h) affixing the product of step (g) to a membrane; (i) treating the membrane under hybridization conditions with a labeled sequence-specific oligonucleotide probe capable of hybridizing with the amplified nucleic acid sequence only if a sequence of the probe is complementary to a region of the amplified sequence; and (j) detecting whether the probe has hybridized to an amplified sequence in the nucleic acid sample.

Claim 34

A process for detecting the presence or absence of at least one nucleotide variation in sequence in one or more nucleic acids contained in a sample, wherein if the nucleic acid is double-­stranded, it consists of two separated complementary strands of equal or unequal length, which process comprises: (a) contacting the sample with four different nucleotide triphosphates and one oligonucleotide primer, for each strand of each nucleic acid suspected of containing said variation(s), wherein each primer is selected to be substantially complementary to different strands 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, said contacting being at a temperature which promotes hybridization of each primer to its complementary nucleic acid strand; (b) contacting the sample, at the same time as or after step (a), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (c) maintaining the reaction 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 nucleic acid suspected of containing said variation(s), an extension product of each primer which is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (d) heating the reaction mixture from step (c) for an effective time and at an effective temperature to separate the primer extension products from the templates on which they were synthesized to produce single-stranded molecules, but not so high as to denature irreversibly the thermostable enzyme; (e) cooling the reaction 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 reaction 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 nucleic acid suspected of containing said variation(s), an extension product of each primer which is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template, wherein steps (d), (e) and (f) are repeated a sufficient number of times to result in detectable amplification of the nucleic acid containing the sequence variation(s), if present, and wherein at least one primer and/or at least one of the four nucleotide triphosphates is labeled with a detectable moiety, and wherein steps (e) and (f) are carried out simultaneously or sequentially; (g) affixing to a membrane a sequence-specific oligonucleotide capable of hybridizing with the amplified nucleic acid sequence only if a sequence of the oligonucleotide is complementary to a region of the amplified sequence; (h) treating the membrane under hybridization conditions with the product of step (f), and (i) detecting whether an amplified sequence in the nucleic acid sample has hybridized to the oligonucleotide affixed to the membrane.

Claim 35

A process for detecting the presence or absence of at least one nucleotide variation in sequence in one or more nucleic acids contained in a sample, wherein if the nucleic acid is double-­stranded, it consists of two separated complementary strands of equal or unequal length, which process comprises: (a) contacting the sample with four different nucleotide triphosphates and one oligonucleotide primer, for each strand of each nucleic acid suspected of containing said variation(s), wherein each primer is selected to be substantially complementary to different strands 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, said contacting being at a temperature which promotes hybridization of each primer to its complementary nucleic acid strand; (b) contacting the sample, at the same time as or after step (a), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (c) maintaining the reaction 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 nucleic acid suspected of containing said variation(s), an extension product of each primer which is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (d) heating the reaction mixture from step (c) for an effective time and at an effective temperature to separate the primer extension products from the templates on which they were synthesized to produce single-stranded molecules, but not so high as to denature irreversibly the thermostable enzyme; (e) cooling the reaction 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 reaction 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 nucleic acid suspected of containing said variation(s), an extension product of each primer which is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template, wherein steps (d), (e) and (f) are repeated a sufficient number of times to result in detectable amplification of the nucleic acid containing the sequence variation(s), if present, and wherein at least one primer and/or at least one of the four nucleotide triphosphates is labeled with a detectable moiety, and wherein steps (e) and (f) are carried out simultaneously or sequentially; (g) affixing to a membrane a sequence-specific oligonucleotide capable of hybridizing with the amplified nucleic acid sequence only if a sequence of the oligonucleotide is complementary to a region of the amplified sequence; (h) treating the membrane under hybridization conditions with the product of step (f), and (i) detecting whether an amplified sequence in the nucleic acid sample has hybridized to the oligonucleotide affixed to the membrane.

Claim 36

A process for detecting the presence or absence of at least one nucleotide variation in sequence in one or more nucleic acids contained in a sample, wherein the nucleic acid(s) each consist of two complementary strands of equal or unequal length, which process comprises: (a) heating the sample in the presence of four different nucleotide triphosphates and one oligonucleotide primer, for each strand of each nucleic acid suspected of containing said variation(s), for an effective time and at an effective temperature to denature each nucleic acid, wherein each primer is selected to be substantially complementary to different strands of each specific nucleic acid, 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) cooling the denatured nucleic acids to a temperature which promotes hybridization of each primer to its complementary nucleic acid strand; (c) contacting the denatured nucleic acids, at the same time as or after step (a) or (b), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (d) maintaining the reaction 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 nucleic acid suspected of containing said variation(s), an extension product of each primer which is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (e) heating the reaction mixture from step (d) for an effective time and at an effective temperature to separate the primer extension products from the templates on which they were synthesized to produce single-stranded molecules, but not so high as to denature irreversibly the enzyme; (f) cooling the reaction 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 reaction 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 nucleic acid suspected of containing said variation(s), an extension product of each primer which is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template, steps (e)-(g) being repeated a sufficient number of times to result in detectable amplification of the nucleic acid containing the sequence variation(s), if present, wherein at least one primer and/or at least one of the four nucleotide triphosphates is labeled with a detectable moiety, and wherein steps (f) and (g) are carried out simultaneously or sequentially; (h) affixing to a membrane a sequence-specific oligonucleotide capable of hybridizing with the amplified nucleic acid sequence only if a sequence of the oligonucleotide is complementary to a region of the amplified sequence; (i) treating the membrane under hybridization conditions with the product of step (g); and (j) detecting whether an amplified sequence in the nucleic acid sample has hybridized to the oligonucleotide affixed to the membrane.

Claim 37

A process for detecting the presence or absence of at least one nucleotide variation in sequence in one or more nucleic acids contained in a sample, wherein the nucleic acid(s) each consist of two complementary strands of equal or unequal length, which process comprises: (a) heating the sample in the presence of four different nucleotide triphosphates and one oligonucleotide primer, for each strand of each nucleic acid suspected of containing said variation(s), for an effective time and at an effective temperature to denature each nucleic acid, wherein each primer is selected to be substantially complementary to different strands of each specific nucleic acid, 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) cooling the denatured nucleic acids to a temperature which promotes hybridization of each primer to its complementary nucleic acid strand; (c) contacting the denatured nucleic acids, at the same time as or after step (a) or (b), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (d) maintaining the reaction 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 nucleic acid suspected of containing said variation(s), an extension product of each primer which is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (e) heating the reaction mixture from step (d) for an effective time and at an effective temperature to separate the primer extension products from the templates on which they were synthesized to produce single-stranded molecules, but not so high as to denature irreversibly the enzyme; (f) cooling the reaction 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 reaction 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 nucleic acid suspected of containing said variation(s), an extension product of each primer which is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template, steps (e)-(g) being repeated a sufficient number of times to result in detectable amplification of the nucleic acid containing the sequence variation(s), if present, wherein at least one primer and/or at least one of the four nucleotide triphosphates is labeled with a detectable moiety, and wherein steps (f) and (g) are carried out simultaneously or sequentially; (h) affixing to a membrane a sequence-specific oligonucleotide capable of hybridizing with the amplified nucleic acid sequence only if a sequence of the oligonucleotide is complementary to a region of the amplified sequence; (i) treating the membrane under hybridization conditions with the product of step (g); and (j) detecting whether an amplified sequence in the nucleic acid sample has hybridized to the oligonucleotide affixed to the membrane.

Claim 38

A process for detecting the presence or absence of at least one nucleotide variation in sequence in one or more nucleic acids contained in a sample, wherein if the nucleic acid is double-­stranded, it consists of two separated complementary strands of equal or unequal length, which process comprises: (a) contacting the sample with four different nucleotide triphosphates and one oligonucleotide primer, for each strand of each nucleic acid suspected of containing said variation(s), wherein each primer is selected to be substantially complementary to different strands 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, said contacting being at a temperature which promotes hybridization of each primer to its complementary nucleic acid strand; (b) contacting the sample, at the same time as or after step (a), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (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 nucleic acid suspected of containing said variation(s), an extension product of each primer which is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (d) heating the mixture from step (c) for an effective time and at an effective temperature to separate the primer extension products from the templates 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 nucleic acid suspected of containing said variation(s), an extension product of each primer which is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template, wherein steps (d), (e) and (f) are repeated a sufficient number of times to result in detectable amplification of the nucleic acid containing the sequence variation(s), if present, and wherein step (e) and (f) are carried out simultaneously or sequentially; (g) affixing to a membrane a labeled sequence-specific oligonucleotide probe capable of hybridizing with the amplified nucleic acid sequence, only if a sequence of the probe is complementary to a region of the amplified sequence; (h) treating the membrane under hybridization conditions with the product of step (f); (i) treating the product of step (h) with a restriction enzyme which will cleave any hybrids formed if the probe and variation(s) to be detected both contain a restriction site recognized by the enzyme; and (j) detecting whether a labeled restriction fragment of the required length is present in the restriction digest to indicate that hybridization occurred.

Claim 39

A process for detecting the presence or absence of at least one nucleotide variation in sequence in one or more nucleic acids contained in a sample, wherein if the nucleic acid is double-­stranded, it consists of two separated complementary strands of equal or unequal length, which process comprises: (a) contacting the sample with four different nucleotide triphosphates and one oligonucleotide primer, for each strand of each nucleic acid suspected of containing said variation(s), wherein each primer is selected to be substantially complementary to different strands 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, said contacting being at a temperature which promotes hybridization of each primer to its complementary nucleic acid strand; (b) contacting the sample, at the same time as or after step (a), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (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 nucleic acid suspected of containing said variation(s), an extension product of each primer which is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (d) heating the mixture from step (c) for an effective time and at an effective temperature to separate the primer extension products from the templates 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 nucleic acid suspected of containing said variation(s), an extension product of each primer which is complementary to each nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template, wherein steps (d), (e) and (f) are repeated a sufficient number of times to result in detectable amplification of the nucleic acid containing the sequence variation(s), if present, and wherein step (e) and (f) are carried out simultaneously or sequentially; (g) affixing to a membrane a labeled sequence-specific oligonucleotide probe capable of hybridizing with the amplified nucleic acid sequence, only if a sequence of the probe is complementary to a region of the amplified sequence; (h) treating the membrane under hybridization conditions with the product of step (f); (i) treating the product of step (h) with a restriction enzyme which will cleave any hybrids formed if the probe and variation(s) to be detected both contain a restriction site recognized by the enzyme; and (j) detecting whether a labeled restriction fragment of the required length is present in the restriction digest to indicate that hybridization occurred.

Claim 40

A process for cloning into a cloning vector one or more specific nucleic acid sequences contained in a nucleic acid or a mixture of nucleic acids, which nucleic acid(s) when double-stranded consist of two separated complementary strands, and which nucleic acid(s) are amplified in quantity before cloning, which process comprises: (a) contacting each nucleic acid with four different nucleotide triphosphates and one oligonucleotide primer for each different specific sequence being amplified, wherein each primer is selected to be substantially complementary to different strands 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 each sequence being amplified or each primer contains a restriction site, said contacting being at a temperature which promotes hybridization of each primer to its complementary nucleic acid strand; (b) contacting each nucleic acid strand, at the same time as or after step (a) or (b), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (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 nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (d) heating the mixture from step (c) for an effective time and at an effective temperature to separate the primer extension products from the templates 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 nucleic acid strand template produced in step (d), but not so high as to separate each extension product from its complementary strand template, steps (d), (e) and (f) being repeated a sufficient number of times to result in detectable amplification of the nucleic acid(s) containing the sequence(s), wherein steps (e) and (f) are carried out simulteneously 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.

Claim 41

A process for cloning into a cloning vector one or more specific nucleic acid sequences contained in a nucleic acid or a mixture of nucleic acids, which nucleic acid(s) when double-stranded consist of two separated complementary strands, and which nucleic acid(s) are amplified in quantity before cloning, which process comprises: (a) contacting each nucleic acid with four different nucleotide triphosphates and one oligonucleotide primer for each different specific sequence being amplified, wherein each primer is selected to be substantially complementary to different strands 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 each sequence being amplified or each primer contains a restriction site, said contacting being at a temperature which promotes hybridization of each primer to its complementary nucleic acid strand; (b) contacting each nucleic acid strand, at the same time as or after step (a) or (b), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (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 nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (d) heating the mixture from step (c) for an effective time and at an effective temperature to separate the primer extension products from the templates 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 nucleic acid strand template produced in step (d), but not so high as to separate each extension product from its complementary strand template, steps (d), (e) and (f) being repeated a sufficient number of times to result in detectable amplification of the nucleic acid(s) containing the sequence(s), wherein steps (e) and (f) are carried out simulteneously 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.

Claim 42

A process for cloning into a cloning vector at least one specific nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids, wherein the nucleic acid(s) consist of two complementary strands of equal or unequal length, and wherein the nucleic acid(s) are amplified in quantity before cloning, which process comprises: (a) heating each nucleic acid in the presence of four different nucleotide triphosphates and one oligonucleotide primer, for each different specific sequence being amplified, for an effective time and at an effective temperature to denature each nucleic acid, wherein each primer is selected to be substantially complementary to different strands 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 each sequence being amplified or each primer contains a restriction site; (b) cooling the denatured nucleic acids to a temperature effective to promote hybridization between each primer and its complementary strand; (c) contacting the denatured nucleic acids, at the same time as or after step (a) or (b), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (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 nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (e) heating the mixture from step (d) for an effective time and at an effective temperature to separate the primer extension products from the templates 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 nucleic acid strand template produced in step (e), but not so high as to separate each extension product from its complementary strand template, steps (e), (f), and (g) being repeated a sufficient number of times to result in detectable amplification of the nucleic acid(s) containing the sequence(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.

Claim 43

A process for cloning into a cloning vector at least one specific nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids, wherein the nucleic acid(s) consist of two complementary strands of equal or unequal length, and wherein the nucleic acid(s) are amplified in quantity before cloning, which process comprises: (a) heating each nucleic acid in the presence of four different nucleotide triphosphates and one oligonucleotide primer, for each different specific sequence being amplified, for an effective time and at an effective temperature to denature each nucleic acid, wherein each primer is selected to be substantially complementary to different strands 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 each sequence being amplified or each primer contains a restriction site; (b) cooling the denatured nucleic acids to a temperature effective to promote hybridization between each primer and its complementary strand; (c) contacting the denatured nucleic acids, at the same time as or after step (a) or (b), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (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 nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (e) heating the mixture from step (d) for an effective time and at an effective temperature to separate the primer extension products from the templates 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 nucleic acid strand template produced in step (e), but not so high as to separate each extension product from its complementary strand template, steps (e), (f), and (g) being repeated a sufficient number of times to result in detectable amplification of the nucleic acid(s) containing the sequence(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.

Claim 44

A process for cloning into a cloning vector one or more specific nucleic acid sequences contained in a nucleic acid or mixture of nucleic acids, which nucleic acid(s) when double-stranded consist of two separated complementary strands of equal or unequal length and which nucleic acid(s) are amplified in quantity before cloning, which process comprises: (a) contacting each nucleic acid with four different nucleotide triphosphates and one oligonucleotide primer for each different specific sequence being amplified, wherein each primer is selected to be substantially complementary to different strands 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, said contacting being at a temperature which promotes hybridization of each primer to its complementary nucleic acid strand; (b) contacting each nucleic acid strand, at the same time as or after step (a) or (b), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (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 nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (d) heating the mixture from step (c) for an effective time and at an effective temperature to separate the primer extension products from the templates 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 nucleic acid strand template produced in step (d), but not so high as to separate each extension product from its complementary strand template, steps (d), (e) and (f) being repeated a sufficient number of times to result in effective amplification of the nucleic acid(s) 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.

Claim 45

A process for cloning into a cloning vector one or more specific nucleic acid sequences contained in a nucleic acid or mixture of nucleic acids, which nucleic acid(s) when double-stranded consist of two separated complementary strands of equal or unequal length and which nucleic acid(s) are amplified in quantity before cloning, which process comprises: (a) contacting each nucleic acid with four different nucleotide triphosphates and one oligonucleotide primer for each different specific sequence being amplified, wherein each primer is selected to be substantially complementary to different strands 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, said contacting being at a temperature which promotes hybridization of each primer to its complementary nucleic acid strand; (b) contacting each nucleic acid strand, at the same time as or after step (a) or (b), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (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 nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (d) heating the mixture from step (c) for an effective time and at an effective temperature to separate the primer extension products from the templates 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 nucleic acid strand template produced in step (d), but not so high as to separate each extension product from its complementary strand template, steps (d), (e) and (f) being repeated a sufficient number of times to result in effective amplification of the nucleic acid(s) 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.

Claim 46

A process for cloning into a cloning vector at least one specific nucleic acid sequence contained in a nucleic acid or mixture of nucleic acids, which nucleic acid(s) consist of two complementary strands of equal or unequal length and which nucleic acid(s) are amplified in quantity before cloning, which process comprises: (a) heating each nucleic acid in the presence of four different nucleotide triphosphates and one oligonucleotide primer for each different specific sequence being amplified, for an effective time and at an effective temperature to denature each nucleic acid, wherein each primer is selected to be substantially complementary to different strands 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) cooling the denatured nucleic acids to a temperature effective to promote hybridization between each primer and its complementary strand; (c) contacting the denatured nucleic acids, at the same time as or after step (a) or (b), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (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 nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (e) heating the mixture from step (d) for an effective time and at an effective temperature to separate the primer extension products from the templates 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 nucleic acid strand template produced in step (e), but not so high as to separate each extension product from its complementary strand template, steps (e), (f) and (g) being repeated a sufficient number of times to result in effective amplification of the nucleic acid(s) containing each sequence for blunt-end 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 amounts to effect the ligation.

Claim 47

A process for cloning into a cloning vector at least one specific nucleic acid sequence contained in a nucleic acid or mixture of nucleic acids, which nucleic acid(s) consist of two complementary strands of equal or unequal length and which nucleic acid(s) are amplified in quantity before cloning, which process comprises: (a) heating each nucleic acid in the presence of four different nucleotide triphosphates and one oligonucleotide primer for each different specific sequence being amplified, for an effective time and at an effective temperature to denature each nucleic acid, wherein each primer is selected to be substantially complementary to different strands 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) cooling the denatured nucleic acids to a temperature effective to promote hybridization between each primer and its complementary strand; (c) contacting the denatured nucleic acids, at the same time as or after step (a) or (b), with a thermostable enzyme which catalyzes combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; (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 nucleic acid strand template, but not so high as to separate each extension product from its complementary strand template; (e) heating the mixture from step (d) for an effective time and at an effective temperature to separate the primer extension products from the templates 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 nucleic acid strand template produced in step (e), but not so high as to separate each extension product from its complementary strand template, steps (e), (f) and (g) being repeated a sufficient number of times to result in effective amplification of the nucleic acid(s) containing each sequence for blunt-end 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 amounts to effect the ligation.

Claim 48

An amplified nucleic acid sequence from a nucleic acid or mixture of nucleic acids comprising multiple copies of said sequence produced by an amplification process of claim 19 or 20.