Microsatellite genotyping using ABI3100 platform
Service: Accurate allele sizing and scoring of microsatellites/DNA fragments
Platform: The ABI PRISM® 3100 Genetic Analyzer is a multi-colour fluorescence based 16 capillary platform
Application: Microsatellites are polymorphic DNA sequences containing a repeated nucleotide sequence eg. CA, CTC or TGGA. The number of repeat units for a given locus may vary resulting in alleles of different lengths. As Microsatellites are polymorphic they can be used as markers for linkage analysis in disease studies.
Contact: Hazel.Platt@manchester.ac.uk
Technology and theory
Microsatellites can be genotyped by flanking the microsatellite region with PCR primers, one of which has a 5' fluorescent label. Amplification produces a pair of fluorescent allelic products that will vary in size according to the number of microsatellite repeat units. A suitable choice of fluorescent labelling enables analysis of multiple loci in the same capillary injection. Using colour and size to distinguish between fragments it is possible to multiplex up to 10 markers in a single capillary.
Detectable fluorescent labels are FAM (recommended-strong label), HEX (weak label), VIC, NED, and PET. Fluorescent labelled DNA fragments alongside an appropriate size standard migrate through polymer filled capillaries past a laser beam which excites them. A spectralphotometer collects and separates the light emitted by the dyes. A CCD camera converts the fluorescence into digital data that is processed by the 3100 data collection software. Data analysis and subsequent allele sizing and scoring is performed using ABI Genescan, Genotyper or Genemapper software.
Sample throughput
384 samples (10 plex) in 12 hours.
Sample requirement
10ng of DNA@5ng/µL per microsatellite.