How does RFLP Analysis work?
RFLP analysis takes advantage that people's DNA varied with respect to the locations of certain restriction sites (Kluftinger, J. and Plunket, R., 2015). In this method the DNA is digested with restriction enzymes and the resulting fragments are separated on a gel. In order to do an RFLP analysis you need to use restriction enzymes. The fragments can be stained or radioactively labelled to identify the pattern and the pattern can be compared to that of others. The restriction enzymes cut at specific sites in the DNA, and this produces fragments of varying length which are dependent on the person's DNA. A person’s DNA varies based on the genetic linkage of the alleles that are being analyzed and typically Mendelian inheritance is seen. The fact that this analysis relies on a large amount of DNA, RFLP analysis is virtually obsolete in forensics. Some of the restriction enzymes that can been used can be seen in figure 1.
Figure 1. The restriction enzymes (RE) that can be used with restriction fragment length polymorphism (RFLP) to digest the DNA being analyzed (Nelson et al., 2013). The arrows indicate where the restriction enzyme cuts in the palindromic sequence. The restriction enzymes that were used in this lab were EcoRI and Pst1.
In this experiment the RFLP analysis was done using the restriction endonucleases (RE’s) EcoRI and Pst1. This digestion with restriction endonuclease produces the fragments of varying length, called restriction fragment length polymorphisms (RFLPs) that were run on a 1% agarose gel. The gel with the RFLPs makes the RFLP profile which is analogous to a fingerprint for a person.
The protocol consists of obtaining a sample of DNA from the crime scene, digesting it with the desired restriction endonucleases and running on a gel with a DNA fragment size standard which is the ladder. The DNA for each of the crime scene and the suspects was mixed with 10µL of RE mix, which contained EcoRI and Pst1, and centrifuged. The digest was then incubated at 37°C for 45 min.
The protocol consists of obtaining a sample of DNA from the crime scene, digesting it with the desired restriction endonucleases and running on a gel with a DNA fragment size standard which is the ladder. The DNA for each of the crime scene and the suspects was mixed with 10µL of RE mix, which contained EcoRI and Pst1, and centrifuged. The digest was then incubated at 37°C for 45 min.