PCR RFLP

Genetic polymorphisms in genes involved in DNA replication and repair are a category of genes where individual polymorphisms may play an important role in the disease process. Recent advances made in PCR technology have significantly simplified the analysis of genetic polymorphisms. After the common sequence variations observed in a population have been determined by DNA sequencing, PCR strategies can be designed to elucidate the particular allele present in each individual sample. For example, glutathione S-transferase (GST) M1 and/or T1 genes have been shown to be deleted in certain individuals. Using a PCR coamplification analysis with beta-globin as an internal standard and a GST-M1+/GST-T1+ positive control, a large number of samples can be rapidly genotyped with respect to GST status.

Similarly, amplification of a region surrounding a naturally occurring restriction enzyme site, followed by digestion with the appropriate enzyme, can allow a rapid genotyping determination to be made. An example of this strategy is the genotyping of four sequence variations of the NAT2 gene by PCR amplification and four separate restriction analyses. If a naturally occurring restriction site does not exist in either of the expected alleles of a gene, a restriction site can usually be engineered by creative primer design.

BioServe has extensive experience in genotyping analysis, and has appropriate positive controls which are included to ensure completion of digestion of PCR fragments and appropriate gel resolution. If genotyping conditions are not established for a particular gene, scientists at BBL can assist in appropriate primer design and/or establishment of the optimum assay conditions.