RFLP assays (snip-SNPs) - Genetic Mapping Resource for C. briggsae
We have developed restriction fragment length polymorphism (RFLP) assays to facilitate inexpensive genetic mapping in C. briggsae.
Identification of snip-SNPs
We screened the latest build of HK104 SNPs for substitutions that altered the recognition sequence of restriction enzymes.
To make this a practical resource, we limited the analysis to 30 restriction enzymes from REBASE
that are known to be reliable and inexpensive. In the end, 25 of these had at least one RFLP assay per chromosome (see table below).
Primer Design and Assay Development
PCR assays were designed to our standard protocols: amplicon sizes of 500 to 1000 bp, primer TM's of 54-56.
In silico fragment analysis of the PCR products was performed to predict band sizes for AF16 and HK104; assays with more than 4 bands in either strain were removed.
Some 3,723 snip-SNPs passed PCR primer design. These spanned 137 cb25 ultra-contigs, 84 of which were in the current genetic map.
Our final set contained 1,987 RFLP assays, now positioned on the "cb3" sequence
assembly, that can be used for genetic mapping in C. briggsae.