Cloning Long Polymerase Chain Reaction Products

A variety of methods have been developed and modified to facilitate the cloning of polymerase chain reaction (PCR) products directly into plasmid. However, there are limitations in practice, including the low frequency of correct clones, the expense of commercially available cloning systems, and the need to add sequence or other modifications to primers to enable cloning. The more recent development of long and accurate PCR (LA-PCR) offers the ability to isolate much longer products. Using this technique, the vast majority of full-length eucaryotic mRNA transcripts can be easily amplified by reverse transcription and LA-PCR. In addition, since the coding region for the majority of mammalian genes is contained within approx 20 kilobases (kb) or less of genomic DNA, this technology can be used to rapidly obtain genomic sequence corresponding to known cDNAs.