Detection of DNA Polymorphisms Using PCR-RFLP and Capillary Electrophoresis

The most fundamental difference between two individuals is DNA variation at the nucleotide level. This DNA variation, often referred to as a single nucleotide polymorphism or SNP, can occur approximately once every several hundred base pairs. The ability to accurately determine the DNA sequence at specific sites throughout an organism’s genome is important in a variety of applications including disease detection, differentiation of microorganisms, agricultural genetics and molecular breeding, and human identification (i.e., forensic and paternity testing). A number of laboratory techniques have been developed in recent years to analyze DNA rapidly and reliably for the purpose of mutation detection. One of these methods involves the use of DNA restriction enzymes to generate DNA molecules of varying length depending on the presence or absence of restriction sites. The length variations in DNA molecules caused when the restriction enzymes do their cutting are called restriction fragment-length polymorphisms (RFLP). Another molecular procedure that allows one to work with small amounts of DNA is the polymerase chain reaction (PCR), a method for amplifying and enriching the sample within a targeted region of a DNA sequence. When the two procedures are combined, the method is referred to as PCR-RFLP. A separation step is required to resolve and measure the length of the restriction fragments from one another after the enzymatic reactions ( Fig. 1 ), and this essential step is where capillary electrophoresis (CE) offers a number of advantages.