Promoter ligation activated transcription amplification of nucleic acid sequences

• US 5,194,370 A
• Filed: 05/16/1990
• Issued: 03/16/1993
• Est. Priority Date: 05/16/1990
• Status: Expired due to Fees

• Current Assignee: Life Technologies Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Berninger, Mark S., Rashtchian, Ayoub, Schuster, David M.
• Primary Examiner(s): Moskowitz, Margaret
• Assistant Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US07/524,306
• Time in Patent Office: 1,035 Days
• Field of Search: 435/6, 435/91, 435/193, 435/194, 435/199, 435/810, 935/77, 935/78, 436/501, 436/94
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

This invention discloses a scheme for producing nucleic acid end products that are functionally or exactly identical to the starting products, thereby resulting in exponential amplification of a desired nucleic acid sequence. Specifically, sequences are cycled between RNA and DNA forms using the following basic steps: (1) a T7 RNA polymerase promoter is ligated onto a single-stranded DNA template; (2) T7 RNA polymerase makes many copies of RNA: (3) a complementary DNA is made from the RNA by extension of a primer by reverse transcriptase; and (4) the RNA template is removed by ribonuclease H. This amplification method is useful for purposes such as genetic research and diagnostic assays.

Claims

1. A method for amplifying and detecting a target DNA sequence present, or possibly present, in a nucleic acid sample in a reaction volume under incubation conditions, comprising:

(a) adding under hybridizing conditions to said sample the following components;

(1) a primer,

(2) a proto-promoter comprising

(a) a double-stranded segment which encodes a promoter for a DNA dependent RNA polymerase, and

(b) a single-stranded segment which is complementary to the 3'"'"' end of the target DNA and which is a 3'"'"' overhand;

(3) ribonucleotide triphosphates,

(4) deoxyribonucleotide triphosphates,

(5) RNA-dependent DNA polymerase,

(6) an enzyme capable of releasing single-stranded DNA from RNA-DNA heteroduplexes,

(7) DNA ligase,

(8) DNA-dependent RNA polymerase, and

(9) buffer solution;

(b) annealing together said proto-promoter and target DNA, thereby forming a target DNA/proto-promoter complex;

(c) ligating together the target DNA/photo-promoter complex, thereby forming a covalently-bound target DNA/proto-promoter combination;

(d) transcribing the covalently-bound target DNA/proto-promoter combination with DNA-dependent RNA polymerase, thereby forming transcribed RNA;

(e) annealing the primer to the transcribed RNA, thereby forming a reverse transcription initiation complex;

(f) reverse transcribing the RNA with RNA-dependent DNA polymerase by extension of the primer of the reverse transcription initiation complex to form additional DNA, thereby forming a DNA-RNA heteroduplex;

(g) releasing the DNA strand of the DNA-RNA heteroduplex, thereby liberating the additional DNA;

(h) repeating step (b) through (g), thereby providing a cyclical process; and

(i) detecting nucleic acids synthesized from said cyclical process steps (b) through (h);

wherein said steps (b) through (h) are carried out without temperature cycling.

3. A method as in claim 1, wherein the releasing of step (g) is digesting the DNA-RNA heteroduplex with ribonuclease H, thereby degrading the RNA of the DNA-RNA heteroduplex and liberating copy DNA.

4. A method as in claim 1, wherein the releasing step (g) is helix unwinding of the DNA-RNA heteroduplex with helicase, thereby denaturing the DNA-RNA heteroduplex and liberating copy DNA.

7. A method as in claim 3 or 5, wherein the ribonuclease H is E. coli ribonuclease H.

8. A method as in claim 1, wherein the target nucleic acid is single-stranded DNA.

9. A method as in claim 1, wherein the target nucleic acid is double-stranded DNA.

10. A method as in claims 1 or 2, wherein the primer is at least 8 nucleotides long.

11. A method as in claims 1 or 2, wherein the single-stranded segment of the proto-promoter is at least 4 nucleotides long.

12. A method as in claims 1 or 2, wherein the single-stranded segment of the proto-promoter is at least 8 nucleotides long.

13. A method as in claims 1 or 2, wherein the double-stranded segment of the proto-promoter is at least 22 nucleotide pairs long.

14. A method as in claims 1 or 2, wherein the RNA-dependent DNA polymerase is avian myeloma virus reverse transcriptase.

15. A method as in claims 1 or 2, wherein the RNA-dependent DNA polymerase is Moloney murine leukemia virus reverse transcriptase.

16. A method as in claim 1 or 2, wherein the DNA ligase is T4 DNA ligase.

17. A method as in claims 1 or 2, wherein the DNA-dependent RNA polymerase is T7 DNA polymerase.

18. A method as in claims 1 or 2, wherein 3'"'"'-end of the single-stranded segment of the proto-promoter is incapable of serving as a primer for extension by the RNA-dependent DNA polymerase.

19. A method as in claims 1 or 2, wherein 3'"'"'-end of the single-stranded segment of the proto-promoter is a cordycepin residue.

2. A method for amplifying and detecting a target RNA sequence present, or possibly present, in a nucleic acid sample in a reaction volume under incubation conditions, comprising:

(a) adding under hybridizing conditions to said sample the following components;

(1) a primer,

(2) a proto-promoter comprising

(a) a double-stranded segment which encodes a promoter for a DNA dependent RNA polymerase, and

(b) a single-stranded segment which is complementary to the 3'"'"' end of copy DNA reverse transcribed from target RNA and which is a 3'"'"' overhang;

(3) ribonucleotide triphosphates,

(4) deoxyribonucleotide triphosphates,

(5) RNA-dependent DNA polymerase,

(6) an enzyme capable of releasing single-stranded DNA from RNA-DNA heteroduplexes,

(7) DNA ligase,

(8) DNA-dependent RNA polymerase, and

(9) buffer solution;

(b) annealing the primer to target RNA, thereby forming a first transcription initiation complex;

(c) reverse transcribing target RNA with RNA-dependent DNA polymerase by extension of the primer of the reverse transcription initiation complex to form copy DNA, thereby forming a DNA-RNA heteroduplex;

(d) releasing the DNA strand of the DNA-RNA heteroduplex, thereby liberating copy DNA;

(e) annealing together said proto-promoter and the copy DNA, thereby forming a copy DNA/proto-promoter complex;

(f) ligating together the copy DNA/proto-promoter complex, thereby forming a covalently bound copy DNA/proto-promoter combination;

(g) transcribing the covalently bound copy DNA/proto-promoter combination with DNA-dependent RNA polymerase, thereby forming additional RNA;

(h) repeating steps (b) through (g), thereby providing a cyclical process; and

(i) detecting nucleic acids synthesized from said cyclical process steps (b) through (h);

wherein said steps (b) through (h) are carried out without temperature cycling.

5. A methods as in claim 2, wherein the releasing step (d) is digesting the DNA-RNA heteroduplex with ribonuclease H, thereby degrading the RNA of the DNA-RNA heteroduplex and liberating copy DNA.

6. A method as in claim 2, wherein the releasing step (d) is helix unwinding of the DNA-RNA heteroduplex with helicase, thereby denaturing the DNA-RNA heteroduplex and liberating copy DNA.