Polymerase chain reaction (PCR) and sequencing

For sequence analysis of transfected genes there are two approaches which can be adopted. One is to derive a consensus sequence of the gene in question and this may, in addition to providing the basic sequence, provide limited information on the presence of variant sequences. In order to provide more extensive data on variant sequences a second approach has to be adopted and that is to subclone the transfected gene (or mRNA) and analyse the sequences of large numbers of the subclones. While there are few obstacles nowadays to deriving a consensus sequence, considerable care has to be exercised in interpreting sequence data derived after subcloning because of the possible introduction of laboratory artefacts.