Ligation amplification

• US 7,727,722 B2
• Filed: 01/08/2007
• Issued: 06/01/2010
• Est. Priority Date: 08/29/2001
• Status: Expired due to Fees

• Current Assignee: General Electric Company
• Original Assignee: General Electric Company
• Inventors: Duthie, Robert Scott, Nelson, John Richard
• Primary Examiner(s): CALAMITA, HEATHER
• Application Number:
  US11/620,804
• Publication Number:
  US 20090081644A1
• Time in Patent Office: 1,240 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• 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 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2521/119 RNA polymerase
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2563/103 luminescence

Abstract

Provided herein are methods and agents for ligation-based exponential ribonucleic acid amplification followed by detection using a nucleic acid polymerization reaction employing terminal-phosphate-labeled nucleotides including three or more phosphates as substrates for nucleic acid polymerase.

Claims

1. A method for producing a labeled polyphosphate using amplified RNA sequences, comprising:

(a) amplifying a target RNA sequence to produce the amplified RNA sequences; and

(b) reacting the amplified RNA sequences with a nucleic acid polymerase in presence of at least one terminal-phosphate-labeled nucleotide to produce the labeled polyphosphate, wherein the labeled polyphosphate comprises three or more phosphate groups.

2. The method of claim 1, further comprising the step (c) of detecting the labeled polyphosphate.

3. The methods of claim 2, wherein the amplification step and the detection step are performed in a single reaction vessel.

4. The methods of claim 2, wherein each of the steps (a), (b), and (c) occur substantially simultaneously.

5. The method of claim 1, wherein the target RNA sequence is hetero-polymeric.

6. The method of claim 1, wherein the amplified RNA sequences are antisense cRNA to the target RNA sequence.

7. The method of claim 1, wherein the amplified RNA sequences are the same sense as the target RNA sequence.

8. The method of claim 1, wherein the reacting step comprises incubation of the amplified RNA sequences with a reverse transcriptase, nucleotides, and a primer.

9. The method of claim 8, wherein the primer is complementary to an internal sequence of the amplified RNA sequences.

10. The method of claim 8, wherein the primer consists of about 10 to about 40 nucleotides

11. The method of claim 8, wherein the nucleotides are structurally modified to be substantially non-reactive to phosphatases.

12. The method of claim 2, wherein the labeled polyphosphate is reacted with a phosphatase to produce a detectable species and detecting the formed detectable species.

13. The method of claim 12, wherein the detectable species is produced in amounts substantially proportional to the amount of the target RNA sequence.

14. The method of claim 2, further comprising the step of quantifying the target RNA sequence.

15. The method of claim 1, wherein the amplification of the target RNA sequence, comprising:

providing the target RNA sequence other than poly A;

providing one or more nucleic acid sequences comprising a double stranded region comprising a promoter sequence for RNA polymerase, and a single stranded 3′

terminal region capable of hybridizing to the target RNA sequence;

hybridizing the single stranded 3′

terminal region of the nucleic acid sequence to the target RNA sequence;

ligating 5′

end of the double stranded region of the nucleic acid sequence to 3′

end of the target RNA sequence to form a ligated sequence; and

transcribing the ligated sequence with RNA polymerase to form amplified antisense, complementary RNA sequences.

16. The method of amplification of claim 15, further comprising:

(a) providing the amplified antisense, complementary RNA sequence;

(b) providing one or more nucleic acid sequences comprising a double stranded region comprising a promoter sequence for RNA polymerase, and a single stranded 3′

terminal region capable of hybridizing to the antisense, complementary RNA sequence;

(c) hybridizing the single stranded 3′

terminal region of the nucleic acid sequence to the antisense, complementary RNA sequence;

(d) ligating 5′

end of the double stranded region of the nucleic acid sequence to 3′

end of the antisense, complementary RNA sequence acid to form a second ligated sequence; and

(e) transcribing the second ligated sequence with RNA polymerase to form amplified RNA sequences having the same sense as the target RNA sequence.

17. The methods of claim 15, wherein the nucleic acid sequences comprise DNA

18. The method of claim 15, wherein recessed 5′

end of the nucleic acid sequences is phosphorylated and the terminal single stranded 3′

end is blocked with a dideoxy extension blocker.

19. The method of claim 15, wherein the single stranded 3′

terminal region of the nucleic acid sequences is at least 3 nucleotides long.

20. The method of claim 15, wherein the double stranded region of the nucleic acid sequences is at least 14 nucleotide pairs long.

21. The method of claim 15, wherein the ligation of step is carried out enzymatically.

22. The method of claim 15, wherein the RNA polymerase is a DNA-dependent RNA polymerase.

23. The method of claim 22, wherein the DNA-dependent RNA polymerase is a T7 RNA polymerase.

24. The method of claim 21, wherein the enzyme is a DNA ligase.

25. The method of claim 24, wherein the DNA ligase is a NAD dependent.