Overview of Exon Resequencing

The availability of the human genome sequence allows a more detailed look at the variation between individuals. The most common type of variations are Single Nucleotide Polymorphisms (SNPs), which account for 90% of all variants, the rest being Insertions or deletions. A subset of sequence variants are coding SNPs (cSNPs). These cSNPs have the potential to alter the coding sequence of a gene and may affect the function. Other variants outside exons may affect gene function, for example mRNA stability.

By amplifying individual exons, using PCR, in a panel of individuals and sequencing the product, it is possible to detect all types of common variants. This can be applied genome wide by resequencing the exons from all known genes. In conjunction with a number of other teams, Team 51, Team 65 and Team 71, we initially aim to sequence the exons from all known genes in a panel of 48 individuals of Caucasian origin. The protocol could easily be adapted to study DNA of other ethnic origin or to study samples collected from individuals with particular diseases or other phenotypes. We will also add other functionally important regions of genes (e.g. promoters, enhancers) to the analysis.

Primers are designed at least 50 basepairs either side of each exon. Initially all exons are amplified at 60oC. After amplification a sample of the products are visualised on a gel, to confirm that products of expected size have been amplified. The remaining product is then cleaned up using two enzymes, Exonuclease 1 and Shrimp Alkaline Phosphatase, which breakdown any remaining primers and dinucleotides, so they don't interfere with the sequencing reactions.

The sequences for each exon are aligned and then the analysis to look for variation can be done. An automatic SNP analysis tool is being developed by Steve Miller and Steve Leonard. Below is an example of a candidate SNP.

The DNA being used in this study from paternal and maternal grandparents from the following CEPH pedigrees;