Cleaved Amplified Polymorphic Sequences (CAPS) polymorphisms are differences in restriction fragment lengths caused by SNPs or INDELs that create or abolish restriction endonuclease recognition sites in PCR amplicons produced by locus-specific oligonucleotide primers.
How It Works
The CAPS assay uses amplified DNA fragments that are digested with a restriction endonuclease to display RFLP.
Unique sequence primers are used to amplify a mapped DNA sequence from two related individuals (for example, from two different inbred ecotypes), A/A and B/B, and from the heterozygote A/B. The amplified fragments from A/A and B/B contain two and three RE recognition sites, respectively. In the case of the heterozygote A/B, two different PCR products will be obtained, one which is cleaved three times and one which is cleaved twice. When fractionated by agarose or acrylamide gel electrophoresis, the PCR products digested by the RE will give readily distinguishable patterns. Some bands will appear as doublets.
- Most CAPS markers are co-dominant and locus-specific.
- Most CAPS genotypes are easily scored and interpreted.
- CAPS markers are easily shared between laboratories.
- CAPS assay does not require the use of radioactive isotopes, and it is more amenable, therefore, to analyses in clinical settings.
Developing CAPS markers
- Sequence the RFLP probe.
- Design primers to amplify 800–2,000-bp DNA fragments. Targeting introns or 3' untranslated regions should increase the chance of finding polymorphisms
- The PCR product is cloned and sequenced.
- PCR amplify DNA fragments from target genotypes, separately digest the amplicons with one or more restriction emzymes.
- Screen the digested amplicons for polymorphism on gels stained with ethidium bromide.