RFLP Lab: Cystic Fibrosis Diagnosis

Keithen B Cast TA: Ben Gamache

Abstract: Introduction: In this lab restriction fragment length polymorphism was used to diagnose as cystic fibrosis patient.Restriction fragment length polymorphism, also known as RFLP, is very useful analysis technique used to identify genetic anomalies that are associated with disease RFLP was the first DNA profiling technique and has largely contributed to DNA fingerprinting, paternity testing, and diagnosis of genetic diseases. RFLP analysis involes electrophoresis of an agarose gel and the use of restriction enzmes for specifically cutting DNA sequences. Restriction enzymes are endonucleases that catalize the cleaving of phosphate bonds through double stranded DNA. These enzymes require Mg+2 for activation. Restriction enzymes produce a 3 hydroxyl group at the point of cleavage and generate a 5 phosphate group. Restriction enzmes are produced by many diffenerent types of bacteria and including blue/green algae. There have been over 1500 restriction enzmes isolated and catalogued for industrial use. The enzyme is named after the organism that it was isolated from using the first few letters of the genus. The difference that distinguishes restriction enzymes from others it site specificity. Restriction enzymes recognize cites from 4-8 nucleotide base pairs long. consequently the enzyme will cut on or near the reconition cit. It is important to note that in any DNA sequence a restriction enzyme cill cut at all the recognition sites. Methods: DNA from a control source and Jeffs DNA were obtained. Four microrocentrifuge tubes were obtained andlabelled Jeff1, Jeff 2, neg 1, and neg 2. After, 10 ul of reaction buffer was added to each of the four tubes. Next 15ul of Jeffs DNA was administered to the microcentrifuge tubes Jeff 1 and Jeff 2. 15 ul of negative control DNA was added to neg 1 and neg 2. In the next step 15 ul of enzyme 1 was added to tubes Jeff 1 and neg 1 and 15 ul of enzyme 2 was added to tubes labelled Jeff 2 and neg 2. Immediately after, the tubes were incubated for 30 minutes at 37 celcius. During incubation the 0.8% agarose gel was prepared. 0.4 grams of agarose was added to 50 ml of 1TAE buffer. The mixture was headed and stired until there were no visable agarose molecules. Ethidium bromide was added to the solution by the TA. The gel was poured into the gel holder and allowed to cool for 20 minutes at room temperature. After cooling the gel was removed and placed in the electrophoresis chamber. the electrophoresis chamber was filled with TAE buffer until the gel was completely submersed. Concluding incubation, 5 ul loading dye was added to each of the four microcentrifuge tubes. A tube of marker DNA, Positive contol DNA cut with enzyme 1, and positive control DNA with enzyme 2 were obtained. All of the microcentrifuge tubes were loaded into the agarose gel to start electrophoresis. The electrophoresis chamber was run at 120 volts for 30-45 minutes. After adequate time, the gels were removed from the chamber and placed in a UV transilluminator for viewing. Results: