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-specific primers. This is a unified method for 5' and 3' rapid amplification of cDNA ends (RACE) from the same adaptor-ligated ds cDNA template. A specially designed adaptor combines features of "vectorette PCR" and "suppression PCR" technologies that significantly reduce background during amplification. The application of "long and accurate PCR" (LA PCR) technology makes possible the amplification of large RACE products and full-length cDNAs with high fidelity to the original mRNA. We investigated efficacy and limitations of this PCR-based approach for cDNA cloning by amplification of 5'- and 3'-RACE fragments and full-length cDNAs of three members of the abundant human actin gene family (1.3-1.9 kb), the medium abundance transferrin receptor mRNA (5.0 kb) and the low-medium abundance insulin-like growth factor II receptor mRNA (9.1 kb).
Chenchik
A
A
CLONTECH Laboratories, Inc., Palo Alto, CA, USA. aachenchik@clontech.com
Diachenko
L
L
Moqadam
F
F
Tarabykin
V
V
Lukyanov
S
S
Siebert
P D
PD
D000199:Actins / Q000145:classification / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000737:chemistry; D005069:Evaluation Studies as Topic; D006801:Humans; D008969:Molecular Sequence Data; D005810:Multigene Family; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics*; D017527:Receptor, IGF Type 2 / Q000235:genetics; D011990:Receptors, Transferrin / Q000235:genetics
D000199:Actins; D018076:DNA, Complementary; D004277:DNA, Single-Stranded; D012333:RNA, Messenger; D017527:Receptor, IGF Type 2; D011990:Receptors, Transferrin