RSCA using ABI3100 platform

Name of service: Reference strand mediated conformation analysis (RSCA) genotyping

Service being provided: MHC class I and class II allele identification in companion animal species, and any other species as requested

Platform used: ABI 3100 capillary electrophoresis

Application of service: Identification of MHC class I and class II alleles & haplotypes

Contact: Lorna.Kennedy@manchester.ac.uk

Technology and theory

Reference Strand-mediated Conformation Analysis (RSCA) is a double stranded conformation-based mutation detection system, which provides a simple, accurate, high throughput technology for the detection of allelic variants and the genotyping of complex polymorphic loci.

RSCA utilises a fluorescein-labelled reference (FLR) DNA molecule, generated by PCR amplification of the locus of interest. The FLR is mixed with locus specific PCR products, denatured and annealed with the test samples.

The nucleotide sequence similarity between the FLR and the unknown sample allows efficient annealing between the sense and anti-sense strands of all the DNA molecules present in the reaction mixture. Since the fluorescein label is present exclusively on the sense strand of the FLR, only duplexes formed with these strands will be detected by the laser detection system of the automated DNA sequencer. Each heteroduplex conformation generated is unique and is influenced by the number and location of the base-pair mismatches. Because each anti-sense strand is different, the mobility of each heteroduplex species is also unique.

A feature distinguishing RSCA from other techniques such as SSCP is the fact that the use of different FLR’s allows the operator to manipulate heteroduplex formation in order to achieve optimal separation of different alleles. The number of different peaks observed will equal the number of alleles present in the sample.

We offer a service to help optimise RSCA for any species, as requested by our customers. To date this method has been used to genotype MHC genes in dogs, cats, horses, bank voles, field voles, caribou, wolves and red jungle fowl.