Method of synthesizing cdna

• US 20060246453A1
• Filed: 03/29/2004
• Published: 11/02/2006
• Est. Priority Date: 03/28/2003
• Status: Active Grant

• Current Assignee: Hitachi High-Tech Company Limited (Hitachi, Ltd.), Japan As Represented By Director General Of National Rehabilitation Center Foor Persons With Disabilities, Seishi Kato
• Original Assignee: Hitachi High-Technologies Corporation (Hitachi, Ltd.)
• Inventors: Kimura, Tomoko, Ohtoko, Kuniyo, Kato, Seishi
• Granted Patent: US 8,067,205 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR

Abstract

A method for synthesizing cDNA possessing a consecutive sequence starting with a nucleotide adjacent to a cap structure of mRNA, which comprises (i) a process for annealing a double-stranded DNA primer and an RNA mixture containing mRNA possessing a cap structure, (ii) a process for preparing a conjugate of an mRNA/cDNA heteroduplex and a double-stranded DNA primer by synthesizing the first-strand cDNA primed with the double-stranded DNA primer using reverse transcriptase, and (iii) a process for circularizing the conjugate of the mRNA/cDNA heteroduplex and the double-stranded DNA primer by joining the 3′ and 5′ ends of the DNA strand containing cDNA using ligase. This method enables us to efficiently synthesize a full-length cDNA possessing a consecutive sequence starting with a transcription-start-site nucleotide from a small amount of RNA by small processes.

Claims

1. A method for synthesizing cDNA possessing a consecutive sequence starting with a nucleotide adjacent to a cap structure of mRNA, which method comprises the steps of:

(i) annealing a double-stranded DNA primer and an RNA mixture containing mRNA possessing a cap structure,

(ii) preparing a conjugate of an mRNA/cDNA heteroduplex and a double-stranded DNA primer by synthesizing the first-strand cDNA primed with the double-stranded DNA primer using reverse transcriptase, and

(iii) circularizing the conjugate of the mRNA/cDNA heteroduplex and the double-stranded DNA primer by joining the 3′ and

5′

ends of the DNA strand containing cDNA using ligase.

2. The method of claim 1, wherein mRNA possessing a cap structure is contained in a cell extract.

3. The method of claim 1, wherein mRNA possessing a cap structure is synthesized by in vitro transcription.

4. The method of claim 1, wherein the primer sequence of the double-stranded DNA primer contains a sequence complementary to a partial sequence of mRNA possessing a cap structure.

5. The method of claim 1, wherein the primer sequence of the double-stranded DNA primer contains an oligo dT complementary to a poly(A) sequence of mRNA possessing a cap structure.

6. The method of claim 1, wherein the ligase is T4 RNA ligase.

7. The method of claim 1, which comprises the following step between the step (ii) and the step (iii):

(ii′

) generating a 5′

-protruding end or a blunt end at the terminal of the double-stranded DNA primer by cutting the conjugate of the mRNA/cDNA heteroduplex and the double-stranded DNA primer using a restriction enzyme.

8. The method of claim 1, which further comprises the following step:

(iv) synthesizing the second-strand cDNA by replacing an RNA strand with a DNA strand in the conjugate of the mRNA/cDNA heteroduplex and the double-stranded DNA primer.

9. The method of claim 8, wherein the double-stranded DNA primer contains a replication origin or both a replication origin and a promoter for cDNA expression.

10. The method of claim 8, which further comprises the following step:

(v) incorporating the double-stranded cDNA composed of the first-strand cDNA and the second-strand cDNA into a vector DNA.

11. A cDNA library that is a population of clones containing double-stranded cDNA synthesized by the method of claim 8, of which more than 60% of the cDNA clones possesses a 5′

-end nucleotide of (dT)ndG (n=0-5) followed by a consecutive sequence starting with a nucleotide adjacent to a cap structure of mRNA.

12. A method for selecting a cDNA clone possessing a consecutive sequence starting with a nucleotide adjacent to a cap structure of mRNA, from clones in the cDNA library of claim 11, wherein a cDNA clone possessing a 5′

-end nucleotide of (dT)ndG (n=0-5) is selected as an objective clone.

17. A cDNA library that is a population of clones containing double-stranded cDNA synthesized by the method of claim 10, of which more than 60% of the cDNA clones possesses a 5′

-end nucleotide of (dT)ndG (n=0-5) followed by a consecutive sequence starting with a nucleotide adjacent to a cap structure of mRNA.

18. A method for selecting a cDNA clone possessing a consecutive sequence starting with a nucleotide adjacent to a cap structure of mRNA, from clones in the cDNA library of claim 17, wherein a cDNA clone possessing a 5′

-end nucleotide of (dT)ndG (n=0-5) is selected as an objective clone.

13. A double-stranded DNA primer possessing an oligo (dT)n (n=15-100) as a primer part, in which one terminal part of a primer side has an 8-base recognition restriction enzyme site RE1, and another terminal part has an 8-base recognition restriction enzyme site RE2 and a restriction enzyme site RE3 generating a 5′

-protruding end or a blunt end.

14. The double-stranded DNA primer of claim 13, which contains a replication origin or both a replication origin and a promoter for cDNA expression.

15. The double-stranded DNA primer of claim 14, which is a vector primer derived from pGCAP10 comprising the nucleotide sequence of SEQ ID NO:

2.

16. A reagent kit for cDNA synthesis, which comprises the double-stranded DNA primer of claim 14, reverse transcriptase and its reaction buffer solution, T4 RNA ligase and its reaction buffer solution, and model mRNA possessing a cap structure.

19. A reagent kit for cDNA synthesis, which comprises the double-stranded DNA primer of claim 15, reverse transcriptase and its reaction buffer solution, T4 RNA ligase and its reaction buffer solution, and model mRNA possessing a cap structure.