Rapid cloning of cDNA ends polymerase chain reaction of G-protein-coupled receptor kinase 6: an improved method to determine 5'- and 3'-cDNA ends.
Rapid cloning of 5'- and 3'-cDNA ends polymerase chain reaction (5'-/3'-RACE-PCR) is useful to determine unknown 5'- and 3'-cDNA termini. Even if the method can yield complete cDNA sequences within a couple of days, the RACE procedure bears some characteristic traps and often results in amplification of unspecific PCR-products. Here we used improved 5'- and 3'-RACE-PCR protocols to obtain the complete cDNA sequence of the G-protein-coupled receptor kinase 6 (GRK6) from a rat brain cDNA library. The use of an anchored oligo-(dT)16-V-primer in the cDNA synthesis, the addition of single-sided PCR steps prior to the RACE-PCRs and the optimization of the dA-tailing reaction conditions in 5'-RACE enhanced RACE-PCR efficiency. Taken together, the method is a tool to determine unknown 5' and 3'-cDNA ends and enables the detection of different transcription initiation sites and mRNA splice variants even from small tissue samples like distinct brain regions. The extensive troubleshooting section discusses typical problems of each substep and contains additional references for support protocols.
Fehr
C
C
Department of Psychiatry, University of Mainz, Germany.
Ficková
M
M
Hiemke
C
C
Dahmen
N
N
D000818:Animals; D001923:Brain Chemistry; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D054768:G-Protein-Coupled Receptor Kinases; D008297:Male; D009419:Nerve Tissue Proteins / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*