Random-primed cDNA synthesis facilitates the isolation of multiple 5'-cDNA ends by RACE

Random-primed cDNA synthesis facilitates the isolation of multiple 5'-cDNA ends by RACE.

The RACE (rapid amplification of cDNA ends) technique (1, 2) can be used to amplify 5'and 3'-cDNA ends that derive from transcripts of low abundance. To isolate the 5' end of a specific cDNA (5' RACE), a small, anti-sense, transcript-specific oligonucleotide is used to prime first-strand cDNA synthesis. The specific first-strand cDNA is then purified, and polyadenylated using terminal deoxynucl...

Rolling circle amplification-RACE: a method for simultaneous isolation of 5' and 3' cDNA ends from amplified cDNA templates.

Directional random oligonucleotide primed (DROP) global amplification of cDNA: its application to subtractive cDNA cloning.

We describe a method of global PCR amplification of cDNA such that the strand sense is maintained. The products of this process are random primed fragments ranging in size from 100 to 500 bp which facilitates uniform PCR amplification of total cDNA. Directional incorporation of a T7 RNA polymerase initiator/promoter sequence allows efficient synthesis of total sense RNA from this material and t...

Full-length cDNA cloning and determination of mRNA 5' and 3' ends by amplification of adaptor-ligated cDNA.

An efficient cDNA amplification procedure is described for determining of the 5' and 3' ends of mRNAs and cloning full-length cDNAs. In this approach, a double-stranded (ds) adaptor is ligated to both ends of a library of ds cDNA by T4 DNA ligase. This adaptor-ligated ds cDNA is then used to selectively amplify 5'- or 3'-cDNA fragments by PCR with a combination of gene-specific and adaptor-spec...

Isolation of a rat liver Golgi mannosidase II clone by mixed oligonucleotide-primed amplification of cDNA.

A clone encoding Golgi mannosidase II (MII; GlcNAc-transferase I-dependent alpha 1,3(alpha 1,6) mannosidase), an enzyme involved in asparagine-linked oligosaccharide processing, was isolated from a rat liver lambda gt11 cDNA library by a method that employs a modification of the polymerase chain reaction. Specific oligonucleotide primers were designed from two regions of protein sequence and we...