Mapping DNA using Restriction Enzymes Ava II and Pvu II to cut Bacterial DNA Abstract
The objective of this project is to map bacterial DNA, which is derived from E. coli, using restriction endonucleases with gel electrophoresis. The DNA fragments, after cutting has occurred, are separated using agarose gel electrophoresis. The DNA fragments are placed in the gel, and an electric current is run through the matrix of the gel-like agarose. Migration of the fragments across the gel is based on the size and charge of the fragment. After the fragments have been run in the gel, they are stained with methylene blue and viewed on a light box. This allows the DNA bands to be viewed on the gel. Introduction
This project outlines the procedure involved in mapping DNA. Prior to mapping, restriction enzymes called restriction endonucleases recognize a specific DNA sequence wherever it occurs in a DNA molecule and cut the DNA at or near the site. Each restriction enzyme is named after the species of bacteria from which it is isolated. Restricting, as it is also known, requires energy in the form of adenosine triphosphate (ATP) and involves physical cleaving of chemical bonds. The sites where cuttings occur are palindromic, that is, the sequences of the complementary strands read the same backward and forward. Naturally, restriction enzymes can recognize and metabolize foreign DNA, and this is of significant importance in bacteria as it constitutes the immune system, since it recognizes and rids it of invaders. After cutting has occurred, gel electrophoresis is used to separate the cut fragments. The gel used in the electrophoresis chamber is agarose gel which is derived from several species of red marine algae, or seaweed. It is also used in size exclusion chromatography. Agarose gel is used to separate and analyze proteins and DNA. In this experiment, agarose gel electrophoresis is used to separate the cut fragments. The DNA fragments are placed in the gel, and an electric current is run through the gel matrix of agarose. It will be observed that the fragments migrate through the gel, but at different rates. It will also be observed that the fragments migrate toward the positive pole. This is due to the negative charge of DNA fragments due to their phosphate backbones. After the DNA samples have been run in the gel, the gel is stained with methylene blue or ethidium bromide which binds with the DNA and fluoresces under ultraviolet light, which allows discrete DNA bands on the gel to be viewed. Gel electrophoresis is important to Biology as it provides data that aids in mapping DNA and determining genetic variation in a population. It is also used to examine tissues found in crime scenes and aid in capturing the criminal. Gel electrophoresis is also used to aid identify damages genes and genetic diseases such as sickle cell anaemia, Huntington’s disease, and Duchenne muscular dystrophy. It is also used to identify viruses. In this experiment, it is hypothesized that during gel electrophoresis, migration of DNA fragments would be to the positive electrode, but at different rates due to their sizes. Therefore, the fragments with smaller sizes will migrate faster than those with larger sizes.
1. Practice pipetting and loading a gel
a. The micropipettor was set to 20 µL.
b. The pipette tip should be attaches to micropipettor. Do not use your hands to attach the tip in order to avoid contamination of the tip. c. Lower the pipette plunger to the first position and carefully place the tip in the practice solution and release gently while the tip is still in the solution. The pipette plunger should be lowered to the second position to dispel the liquid. Do not withdraw liquids by lowering the plunger fully. d. Practice loading the gel. The micropipettor should be held with two hands; one hand should deliver the liquid while the other stabilizes the end. One should make sure that...