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RESEARCH PRODUCT

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

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Abstract 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. Themes: Cell biology, neurotransmitters, modulators, transporters, and receptors Topics: Gene structure and function: general, second messengers and phosphorylation