Title: DNA analysis
Aim: a) Isolate and Purify Bacterial Chromosomal DNA from a strain of E.coli
b) Visualization of restriction fragments by Agarose Gel electrophoresis
* to isolate and purify bacterial chromosomal DNA from a strain of E.coli * to analyze and identify DNA by use of a spectro-photometer * to use restriction enzymes to cleave DNA into fragments * to visualize the restriction fragments by gel electrophoresis * to compare the different DNA fragments generated by use of molecular markers
This work describes a lysis method for the isolation and purification of bacterial genomic DNA and visualization of the restriction fragments by agarose gel electrophoresis. It was noted that for one to isolate and purify bacterial chromosomal DNA several steps are taken into consideration. DNA was found to absorb at 260nm wavelength in a UV spectrophotometer. Restriction enzymes were added to cleave DNA which would produce various DNA fragments. DNA can be separated into different sized fragments by gel electrophoresis. The bacterial DNA was successfully isolated and purified however it could not be observed after running the gel.
DNA analysis is a standard practice for defining paternity or maternity, predisposition to disease, embryonic health and criminal guilty. But in our context, DNA analysis is mainly used for predisposition of diseases in bacteria. Bacteria are pathogenic microorganisms that cause infectious diseases including cholera, syphilis, anthrax and leprosy. The most common fatal bacterial diseases are respiratory infections such as tuberculosis (Barnum S.R; 1998). Nucleic acids encode information relating to cell structure and function. Cells have the ability to make copies of their DNA and pass this information to daughter cells. Nucleic acids are polymers of nucleotides. Nucleotides are composed of ribose (a 5` carbon) sugar and either a purine and pyrimidine base at 1` position. The purine bases are adenine (A) and guanine (G) and the pyrimidine bases are cytosine (C), thymine (T) and Uracil (U). Uracil is only found in RNA and thymine is only found in DNA (Wiser M.F; 2002). Isolation of nucleic acid – three major types of techniques are employed in the isolation of nucleic acids differential solubility, absorption methods or density gradient centrifugation. The choice of method will depend on the type of DNA being isolated and the application. A major goal of nucleic acid isolation is the removal of proteins. The separation of nucleic acids from proteins is generally accomplished due to their different chemical properties. In particular, the highly charged phosphate backbone makes the nucleic acids rather hydrophilic as compared to proteins which are more hydrophobic (Allison L.A; 2012). Spectrophotometry is a versatile analytical tool. The underlying principle of spectrophotometry is to shine light on a sample and to analyze how the sample affects the light. DNA absorbs light at a wavelength of approximately 260nm (Stryer; 2006). Centrifugation is a process that involves the use of the centrifugal force for the separation of mixtures. Separation is based size, shape and density. It utilizes density difference between the particles/macromolecules and the medium in which these are dispersed (Gupta P.K; 2006). Dispersed systems are subjected to artificially induced gravitational fields. A buffer is an aqueous solution consisting of a mixture of weak acid and its conjugate base or weak base and its conjugate acid. Its pH changes very little when a small amount of strong acid or base is added to it and thus it is used to prevent any change in the pH of a solution (Cowan M.K; 2009). Electrophoresis is a diverse technique of separation used to separate and sometimes purify macromolecules especially proteins and nucleic acids that differ in size, charge or conformation by an electric current (Stryer L.; 2006). Gel electrophoresis refers to using a...
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