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

• US 4,965,188 A
• Filed: 06/17/1987
• Issued: 10/23/1990
• Est. Priority Date: 08/22/1986
• Status: Expired due to Term

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Cetus Corporation (Bayer AG)
• Inventors: Gelfand, David H., Erlich, Henry A., Mullis, Kary B., Saiki, Randall K., Horn, Glenn
• Primary Examiner(s): Martinell, James
• Application Number:
  US07/063,647
• Time in Patent Office: 1,224 Days
• Field of Search: 435/6, 435/91, 435/172.3, 435/172.1, 435/320, 435/69.1, 536/27, 935/17, 935/18, 935/76, 935/77, 935/78, 436/63, 436/94, 436/501, 436/508
• US Class Current: 435/6.12
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions

Abstract

A process for amplifying any target nucleic acid sequence contained in a nucleic acid or mixture thereof comprises treating separate complementary strands of the nucleic acid with a molar excess of two oligonucleotide primers and extending the primers with a thermostable enzyme to form complementary primer extension products which act as templates for synthesizing the desired nucleic acid sequence. The amplified sequence can be readily detected. The steps of the reaction can be repeated as often as desired and involve temperature cycling to effect hybridization, promotion of activity of the enzyme, and denaturation of the hybrids formed.

Claims

1. A process for amplifying at least one specific DNA sequence contained in a DNA or a mixture of nucleic acids, wherein if the DNA is double-stranded, it consists of two separated complementary strands of equal or unequal length, which process comprises:

(a) contacting the 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, 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), with 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) 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 strand produced in step (d), but not so high as to separate each extension product from its complementary strand, wherein steps (e) and (f) are conducted simultaneously or sequentially.

2. The process of claim 1, wherein one specific DNA acid sequence is amplified and two primers are employed.

3. The process of claim 1, wherein after steps (d)-(f) are repeated at least once, a second set of two primers is added, wherein the primers added are sufficiently complementary to different strands at internal sequences of the amplified 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.

4. The process of claim 1, wherein steps (d), (e) and (f) are repeated at least five times.

5. The process of claim 4, wherein aid thermostable enzyme is a polymerase from Thermus aquaticus.

6. The process of claim 3 wherein in step (a) the triphosphates and primer(s) are contained in a buffer comprising 1.5-2 mM of a magnesium salt, 150-200 μ

M each of the triphosphates, and 1 μ

M 58°

C., and step (d) is carried out at about 90-100°

C.

7. The process of claim 1, wherein said primers are oligodeoxyribonucleotides.

8. The process of claim 1, wherein said DNA is cDNA.

9. The process of claim 1, wherein the heating and cooling steps (d)-(f) are automated by a machine which controls temperature levels, transitions from one temperature to another, and the timing of temperature levels.

10. The process of claim 1, wherein each primer is present in a molar ratio of at least 1000:

1 primer;

complementary strand.

11. The process of claim 1, wherein at least one primer contains at least one nucleotide which is not complementary to the specific sequence to be amplified.

12. The process of claim 11, wherein said primer that contains at least one nucleotide which is not complementary to the specific sequence to be amplified encodes a promoter.

13. The process of claim 1, wherein steps (e) and (f) are carried out sequentially, steps (c) and (f) take place at 40-80°

C., step (d) takes place at 90-105°

C., and step (e) takes place at 35-65°

C.

14. The process of claim 1 wherein steps (e) and (f) are carried out sequentially, steps (c) and (f) take place at 50-75°

C., step (d) takes place at 90-100°

C., and step (e) takes place at 37°

C.-60°

C.

15. The process of claim 1 wherein steps (e) and (f) are carried out simultaneously at about 45-70°

C.

16. The process of claim 1, wherein said specific DNA sequence is contained in a mixture of nucleic acids.

17. The process of claim 16, wherein said specific DNA sequence is contained in a larger sequence.

18. The process of claim 1, wherein said specific DNA sequence is contained in a larger sequence.

19. A process for detecting the presence of a specific DNA sequence in a sample, said process comprising:

(a) amplifying said sequence by the process of claim 1; and

(b) determining if amplification has occurred.

20. 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.

21. The process of claim 20, wherein said specific DNA sequence is contained in a mixture of nucleic acids.

22. The process of claim 21, wherein said specific DNA sequence is contained in a

23. The process of claim 20, wherein said specific DNA sequence is contained in a larger sequence.

24. A process for detecting the presence of a specific DNA sequence in a sample, said process comprising:

(a) amplifying said sequence by the process of claim 20, and

(b) determining if amplification has occurred.

25. 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.

26. 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.

27. The process of claim 26, wherein one specific DNA sequence is being detected and two primers are employed, and before step (a) the DNA is extracted from the sample.

28. The process of claim 26, wherein steps (d), (e) and (f) are repeated at least 20 times.

29. The process of claim 26, wherein said thermostable enzyme is a polymerase from Thermus aquaticus.

30. The process of claim 26, wherein said primers are oligodeoxyribonucleotides.

31. The process of claim 26, wherein said DNA is cDNA.

32. The process of claim 26, wherein the heating and cooling steps (e)-(g) are automated by a machine which controls temperature levels, transitions from one temperature to another, and the timing of temperature levels.

33. The process of claim 26, wherein the specific DNA sequence to be detected can cause a genetic, infectious or cancerous disease.

34. The process of claim 33, wherein the genetic disease is sickle cell anemia or hemoglobin C disease.

35. The process of claim 34, wherein after step (g) and before step (h) the sample is cut with a restriction enzyme and electrophoresed, and step (i) is accomplished by Southern blot analysis.

36. 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.

46. The process of claim 36, wherein each primer contains a restriction site which is the same as or different from the restriction site(s) on the other primer(s).

37. 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.

39. The process of claim 37, further comprising the step of passing the restriction digest of step (h) through a desalting column or membrane before step (i).

40. The process of claim 37, further comprising, after step (i), sequencing the cleaved product ligated into the vector.

41. The process of claim 37, wherein said specific DNA sequence encodes a protein and wherein said method further comprises, after step (i), expressing the protein encoded by the specific nucleic acid sequence.

42. The process of claim 37, wherein one specific sequence is being amplified, the restriction sites are different on each primer, and the product of step (h) is ligated into one cloning vector with a specific orientation.

43. The process of claim 42, wherein the specific DNA sequence amplified is or is contained within the β

-globin gene or the N-RAS oncogene.

44. The process of claim 37, wherein the thermostable enzyme is a polymerase from Thermus aquaticus.

45. The process of claim 37, wherein the heating and cooling steps (e)-(g) are automated by a machine which controls temperature levels, transitions from one temperature to another, and the timing of the temperature levels.

38. The process of clam 37, wherein steps (e)-(g) are repeated at least five times.

47. The process of clam 46, wherein the restriction sites are on the 5'"'"' end of the primers.

48. 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.

49. 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.

50. The process of claim 49, wherein one DNA sequence is cloned into one vector and two primers are employed.