Dna Digestion and Electrophoresis Essay Example for Free

Dna Digestion and Electrophoresis Essay In this experiment we will be doing a process called as DNA digestion or also known as restriction digest. A restriction digest is a procedure used in molecular biology to prepare DNA for analysis or other processing. It is sometimes termed DNA fragmentation, scientists Hartl and Jones describe it this way: This enzymatic technique can be used for cleaving DNA molecules at specific sites, ensuring that all DNA fragments that contain a particular sequence have the same size; furthermore, each fragment that contains the desired sequence has the sequence located at exactly the same position within the fragment. The cleavage method makes use of an important class of DNA-cleaving enzymes isolated primarily from bacteria. These enzymes are called restriction endonucleases or restriction enzymes, and they are able to cleave DNA molecules at the positions at which particular short sequences of bases are present. The resulting digested DNA is very often selectively amplified using PCR, making it more suitable for analytical techniques such as agarose gel electrophoresis, andchromatography. It is used in genetic fingerprinting, and RFLP analysis. [1] Just as mentioned above, for this experiment we will be using restriction enzymes. Restriction enzymes or restriction endonuclease are enzymes isolated from bacteria that recognize specific sequences in DNA and then cut the DNA to produce fragments, called restriction fragments. They play a very important role in the construction of recombinant DNA molecules, as is done in gene cloning experiments. [2] Restriction endonucleases such as EcoRI recognize specific palindromic sequences and cleave a phosphodiester bond on each strand at that sequence. After digestion with a restriction endonuclease the resulting DNA fragments can be separated by agarose gel electrophoresis and their size can be estimated. A restriction map is generated by using the fragment size data to determine the location of the specific endonuclease recognition sequences on the plasmid. Each restriction enzyme requires specific reaction conditions for optimum activity. One of the most important reaction conditions which varies between different restriction enzymes is the salt concentration. Enzyme buffers are specifically formulated to provide the salt concentration for optimal enzyme activity. It is important, therefore, that the correct buffer solution is used for a particular restriction enzyme. [3] For this experiment we also made use of agarose gel electrophoresis, which takes a lot of time. Electrophoresis may be the main technique for molecular separation in todays cell biology laboratory. In spite of the many physical arrangments for the apparatus, and regardless of the medium through which molecules are allowed to migrate, all electrophoretic separations depend upon the charge distribution of the molecules being separated. Electrophoresis can be one dimensional or two dimensional. One dimensional electrophoresis is used for most routine protein and nucleic acid separations. Two dimensional separation of proteins is used for finger printing , and when properly constructed can be extremely accurate in resolving all of the proteins present within a cell. The support medium for electrophoresis can be formed into a gel within a tube or it can be layered into flat sheets. The tubes are used for easy one dimensional separations, while the sheets have a larger surface area and are better for two- dimensional separations. In electrophoresis, proteins are separated on the basis of charge, and the charge of a protein can be either + or , depending upon the pH of the buffer. In normal operation, a column of gel is partitioned into three sections, known as the Separating or Running Gel, the Stacking Gel and the Sample Gel. The sample gel may be eliminated and the sample introduced via a dense non-convective medium such as sucrose. Electrodes are attached to the ends of the column and an electric current passed through the partitioned gels. If the electrodes are arranged in such a way that the upper bath is (cathode), while the lower bath is + (anode), and anions are allowed to flow toward the anode, the system is known as an anionic system. Flow in the opposite direction, with + cations flowing to the cathode is a cationic system. [4] 1. http://en.wikipedia.org/wiki/Restriction_digest 2. http://www.phschool.com/science/biology_place/biocoach/red/intro.html 3. http://csm.jmu.edu/biology/courses/bio480_580/mblab/restrict.html 4. http://homepages.gac.edu/~cellab/chpts/chpt4/intro4.html