How to use the dCAPS Finder 2.0 program:
- Type or paste the two haplotypes, with no gaps in the sequence, into
the boxes provided. The two sequences must be identical except for the
SNP. The SNP should be in the middle of the sequence with approximately 25
nucleotides on each side. No more than 60 nucleotides should be entered in
either box. A,C, G and T are the only valid characters that will be
accepted by the program.
- In the box provided, enter the number of mismatches allowed in your
PCR primer and run the program. The output from zero mismatches will show
whether a CAPS marker is present. If a CAPS marker is not generated, enter
1 mismatch to search for a dCAPS marker. Increase the number of mismatches
in each run until a potential dCAPS marker has been identified. The dCAPS
primer should include the necessary mismatches 5` of the mutation and not
include the SNP being analyzed.
- The reverse primer should be approximately 200 to 300 nucleotides 3`
of the dCAPS primer such that the two haplotypes can be resolved, after
digestion with the appropriate restriction endonuclease, on a
high-resolution agarose or DNA-agar gel.