Extraction and Characterization of Bacterial DNA

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ABSTRACT:

In this experiment, deoxyribonucleic acid (DNA) was extracted from Micrococcus lysodeikticus, a bacteria with a high guanine and cytosine content. The standard method of chloroform-isoamyl alcohol extraction was used, and the DNA was solubilized in Tris buffer. The DNA was then quantified and qualified using UV spectrophotometry.

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The DNA was determined to have been extracted in relatively high amounts, but the purity was lacking, especially in regards to ribonucleic acid contamination. The hyperchromic effect was utilized to gauge the purity of the DNA. Future studies will focus on the ecologically safer and more efficient methods of DNA extraction.

Key Words: Ribonuclease, RNA, DNA, DNA structure, bacteria. Deoxyribonucleic acid (DNA) is the genetic material inside of a cell. The code of nucleotide base pair sequences allows for the cell to translate messages into proteins that can be used in all parts of the cell. DNA is a double helix that is composed of rungs being the base pairs that are hydrogen bonded together, and a sugar phosphate backbone that is covalently bound together1. This structure gives the DNA the strength yet flexibility it needs in order to be able to unzip so that the genetic material it holds can be accessed2.

The complementarity of the DNA molecule is truly what sets the molecule apart from others. Even if the DNA is denatured and the hydrogen bonds come undone, because each base pair binds to only one specific type of other base pair, this phenomena allows the DNA to spontaneously come together again. The base pairs will line up with their respective pair due to the energetic favorability of having as many hydrogen bonds as possible2.

Because of DNA’s importance as a biological storage molecule, accessing the DNA trapped inside of the cells has become a main avenue of research for scientists. Depending on the organism, different solvents and methods can be used to yield the highest amounts and most pure DNA.

Even within the same types of organisms, there may be different challenges that arise from a specific species. Specifically in bacteria, different strains can have higher or lower G+C content3. Because the guanine and cytosine nucleotides are bound together by three hydrogen bonds as opposed to the 2 that bind adenine and thymine together, the G+C bonds are more difficult to pull apart. This means that harsher solvents may have to be used in order to adequately extract and solubilize the DNA.

When the DNA is extracted, it can be denatured as a way to assess its purity. The hyperchromic effect is a phenomena that occurs when DNA is denatured so that the two strands of the double helix come apart and assume a formation that is random and coiled4. Because there is much more surface area in the randomly coiled DNA than the uniform and compact double helix form,

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