Using Polymerase Chain Reaction-Restriction Length Fragment Polymorphism (PCR-RFLP) method to establish Genetic Variation in Local Mushrooms and Flies

Posted May 12th, 2010 at 1:32 pm.

Clarah Chelagat Lelei
Mentor: Dr. Susan White

Restriction Fragment Length Polymorphism (RFLP) is a variation in the DNA sequence in a genome. In PCR-RFLP technique restriction enzymes plays an important role in DNA cleavage. Restriction enzymes recognize a particular short segment of double stranded DNA that consists of 4 to 12 base pairs. These sites are called restriction sites and they vary in different species because it can occur in the middle of a DNA strand or at the ends. The sites are also palindromic- that is the 5’ to 3’ sequence on one strand is identical to the 5’ to 3’ sequence on the antiparallel complementary strand. The segments resulting from restriction digest vary in size and thus can be detected using gel electrophoresis. For example, HinfI restriction enzyme recognition site is 5’TCGA…3’, HaeIII recognizes 5’GGCC…3’ and Taqα1 recognizes 5’…GANTC…3’ The length of the cleaved DNA varies, and the variation can be detected by performing a gel electrophoresis. The aim of the research is to find out if PCR-RFLP can be used to explain the diversity in the local mushrooms and flies based on the DNA sequence.

My summer research will involve extraction of DNA from local mushroom species and flies. I will then amplify the DNA I have extracted using the Polymerase chain reaction. Specific regions of the DNA will be amplified by thermostat Taq polymerase. A pair of single stranded DNA will act as replication primers and delimits the region of the target molecule that will be amplified. The amplification will be followed by a restriction digest using different restriction enzymes. Then the agarose gel electrophoresis will be performed to visualize the differences in the resulting restriction digest.

If the PCR-RFLP method works, the results from this experiment will help in exploring the genetic variation in mushrooms. The results might also help in explaining why some mushrooms are edible and why some are poisonous and why some insects harmful. The method might also be applied to other species and this may help in answering questions concerning molecular diversity.

Filed under: 2008,Lelei, Clarah Chelagat,White, Dr. Susan by Ann Dixon

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