Method of synthesizing cDNA
View Patent ↗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.
1. A method for constructing a DNA vector having a cDNA synthesized from an mRNA, which method comprises the steps of:
(i) annealing a double-stranded DNA primer and an mRNA mixture, wherein the double-stranded DNA primer consists of a first strand having a primer sequence and a second strand, and wherein the double-stranded DNA primer contains a replication origin or both a replication origin and a promoter for cDNA expression,
(ii) preparing an mRNA/cDNA heteroduplex by synthesizing a first-strand cDNA primed with the primer sequence of the first strand of the double-stranded DNA primer using reverse transcriptase,
(iii) circularizing the mRNA/cDNA heteroduplex by ligating the 3′ end of the first strand cDNA to the 5′ end of the first strand of the double-stranded DNA primer using T4 RNA ligase to form a circular mRNA/cDNA heteroduplex, and
(iv) replacing the RNA in the mRNA/cDNA heteroduplex with a second-strand cDNA by synthesizing the second-strand cDNA with a DNA polymerase, thereby constructing the DNA vector having cDNA consisting of the first-strand cDNA and the second-strand cDNA.
2. The method of claim 1 , wherein the mRNA is contained in a cell extract.
3. The method of claim 1 , wherein the mRNA 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 the mRNA.
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 the mRNA.
6. 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 mRNA/cDNA heteroduplex using a restriction enzyme.