Dna And Restriction Enzyme Essay Research Paper

Dna And Restriction Enzyme Essay, Research Paper

DNA and Restriction Enzyme Interaction

:Identification Of An Unknown Plasmid

Abstract

The fundamental purpose of these experiments is to research the interaction between DNA molecules and restriction enzymes. Two different experiments are performed. In the first experiment, two restriction enzymes, Hind III and Bam H1 interact with an unknown plasmid and the resulting DNA fragments are separated through electrophoresis. Lambda sample is used to create a standard curve, and through it, the sizes of DNA fragments are determined. An uncut sample is used as a negative control, and is compared with cut samples to verify the enzyme activities. The size of the unknown sample is determined to be 4,200 base pairs through the single cut sample. In the double cut sample, the sizes of fragments are determined to be 2,350 and 1,850, which concludes that the unknown sample is pKAN. In the transformation experiment, TE, pAMP, pKAN, and two unknowns are put in five different tubes. E-coli is used as the host bacterium. The TE serves as a negative control because is does not contain DNA. The pAMP and pKAN serve as positive controls and colonize in ampicilin and kanamycin media respectively. The orange-labeled unknown shows identical phenotype with pKAN thus its genotype confirmed to be pKAN. The green-labeled unknown shows identical phenotype with pAMP thus its genotype confirmed to be pAMP.

Introduction

Modern genetic engineering began in 1973 when Herbert Boyer and Stanley Cohen used enzymes to cut a bacteria plasmid – ring of “extra” DNA found outside the nucleus in many single-celled organisms; the relaxed control plasmid was used in experiments because it produced a lot more copies of plasmid than stringent control plasmid1 – and insert another strand of DNA in the gap2. Both bits of DNA were from the same type of bacteria, but this milestone, the invention of recombinant DNA technology, offered a window into the previously impossible — the mixing of traits between totally dissimilar organisms. To prove that this was possible, Cohen and Boyer used the same process to put a bit of frog DNA into bacteria2. In 1990, a young child with an extremely poor immune system received genetic therapy. Some of her white blood cells were genetically manipulated and re-introduced into her bloodstream. These new cells have taken over for the original, weak white cells, and her immune system now works properly2. Although relatively few people have had their cells genetically altered, these advances have made the prospect of mainstream genetic medicine seem more likely.

DNA electrophoreses is a process in which a DNA strand is cut by a restriction enzyme at a certain point so it can be measured and compared with other DNA2. Electrophoresis begins with the pouring of the gel. The gel is an auger poured into a water bath. Combs are then placed in the forming gel to produce wells where the samples are then placed. The gel is placed in the electrophoresis apparatus and loaded with the samples. The different samples are loaded into the wells formed in the gel. The DNA samples are loaded using a measuring instrument called a micropipeter. There is then an electric current passed through the gel to separate the DNA fragments. The current separates the DNA segments by moving the smaller ones farther than the large ones. Every DNA sample separates differently. This way they can be compared with others. It is highly unlikely that two samples will have the same results4.

For about 50 years, antibiotics have been the answer to many bacterial infections. Antibiotics are chemical substances that are secreted by living things. Doctors prescribed these medicines to cure many diseases. During World War II, it treated one of the biggest killers during wartime – infected wounds3. It was the beginning of the antibiotic era. But just when antibiotics were being mass produced, bacteria started to evolve and became resistant to these medicines3.

Bacterium uses the Restriction enzyme as a form of defense mechanism3. Restriction endonuclease, a protein produced by bacteria that cleaves DNA at specific sites along the molecule. It is used to defend against bacterial viruses called bacteriophages, or phages. A phage infects a bacterium by inserting its DNA into the bacterial cells so that it might be replicated. The Restriction enzyme prevents replication of the phage DNA by cutting it into many pieces. In the bacterial cell, Restriction enzymes cleave foreign DNA, thus eliminating infecting organisms. Restriction enzymes can be isolated from bacterial cells and used in the laboratory to manipulate fragments of DNA, such as those that contain genes; for this reason they are indispensable tools of recombinant DNA technology2.

Restriction enzymes recognize certain site (base pair) of an invading foreign phase DNA molecules. If the hypothesis is true, then certain restriction enzymes such as Hind III and Bam H1 will digest certain site of DNA molecules when exposed with foreign phages like pAMP – ampicilin plasmid – and pKAN – kanamycin plasmid. Using given information by the plasmid map cut sizes on p.142 in the Lab manual, if the certain phage is pAMP (4539 base pairs), Hind III will cut the pAMP DNA at the specific site of 1904 base pairs from the origin and Bam H1 will cut the site of 1120 base pairs. If