Dog Genetics Introduction pt 2

In order to gain large qualities of the DNA of interest, DNA and primer amplification is necessary. DNA and primer sequences can be amplified through polymerase chain reaction. In order to look at a particular SNPs, restriction enzymes for that SNP should be amplified using PCR-restriction fragment length, RFLP, also called cleaved amplified polymorphic sequencing, CAP. In the case that there is not a specific digestion enzyme that exists for a particular trait of interest, there is a way to create one. These enzymes are called derived cleaved amplified polymorphic sequences, or dCAPS. These reactions cut and amplify a specific single polymorphism (SNP). A dCAP is used on a mismatched PCR primer to make or get rid of a restriction site based on the particular genotype of the SNP being looked at. The primer used to create a dCAP is mismatched to create the restriction site of interest and the differing alleles are mutated. Primers used do not overlap or mutate the SNP. The restriction enzyme will cut at one site, so if the DNA sequence is cut into two fragments then both strands of DNA contain the allele. However, in the case that the individual is heterozygous, the restriction enzyme will cut only on one strand of DNA resulting in three strands of DNA. Additionally, if the DNA is homozygous for the other allele, there will be no cuts made (J. Laney, dCAPS Primer Design PowerPoint. Moodle.1, 1-16 (2018)).