The newest service from the Genomics Core Facility is primer extension analysis. This is an electrophoresis-only service using researcher-generated primer extension products and LIZ size standards on our ABI 3130 Capillary Electrophoresis Genetic Analyzer. Since the standard is labeled with the fifth dye, users can genotype a greater number of markers in a given capillary, compared to the four-dye system.
Supported Fluorescent Labels
The ABI 3130, using Dye Set G5, will detect fragments labelled with the fluorescent dyes FAM (blue), VIC (green), NED (yellow) and PET (red). The fluorescent dye HEX may be used in place of VIC, and TAMARA may be used in place of NED.
Commonly the LIZ 500 standard is used, allowing for size determinations fom 50 - 500 base pairs.
The LIZ size standard, available from Applied Biosystems, is available in different kits ranging from 120 – 1200 base pairs.
In this chromatogram of the LIZ500, note the asterick above the 250 base pair standard peak. According to ABI, this peak is sensitive to small temperature variations on capillary electrophoresis instruments, and should not be used. Also, primer peaks may interfere with the proper detection of the 35 base pair standard. Be sure to take this information into consideration when optimizing and analyzing your results.
It is the responsibility of the researcher to optimize the primer extension. A good way to this is to run serial dilutions of the primer extension product. In general, dilutions of 1:10 to 1:50 are necessary.
The data may be viewed in STRand or in Peak Scanner from ABI.