ABRF tutorial 2003 update

FARG Tutorial 2003Part 2Single Base Extension: A Method for Detecting Multiple SNPs Using Automated DNA SequencersRebecca Scholl – University of UtahObjective• Discuss the the basics of Single Base Primer Extension• Primer selection ®• Examples using the ABI PrismSNaPshot™ ddNTP Primer Extension Kit• Chemistry and cleanup methods• Sample electrophoresis: Gels v. CapsSingle Base Extension: Advantages• Base discrimination at the target SNP– Homozygous and Hetrozygous status• Can be cheaper than conventional sequencing for multiple SNP sites• Sense and anti-sense strands can be evaluated simultaneously for confirmation of SNP• Can be run on any conventional DNA sequencer available in most labs.– No new equipment costs– Sequencing dyes used• Allows verification of SNP before high throughput screening®SNaPshot Overview3 Step Process– Amplify PCR fragment containing the SNP of interest• Unlabeled primers– Perform primer extension• sense or anti-sense primers• Fluorescent ddNTP incorporated– Visualization of SNPTemplate Selection:• Amplification of a PCR fragment containing the SNP site– Create any sized PCR fragment• 30 - >500 base pairs– Same primers can be used for both amplification steps as long as the primers are specific to the region of interest– Multiple SNPs can be contained in one large PCR template for primer extension• Plasmid Templates– Do not require cleanup before primer extension™Primer Selection: SNaPshot ...