PCR Gel Electrophoresis

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PCR Gel Electrophoresis Introduction Sickle cell anaemia is caused by a Single Nucleotide Polymorphism (SNP). SNPs are the most common type of genetic variation, with each SNP representing a difference in a nucleotide. SNPs occur normally throughout a person’s DNA. Most commonly, they are usually found in non-coding regions and have no effect on health. When a SNP occurs in a coding region, it may play a direct role in disease by affecting the gene’s function. This SNP for sickle cell anaemia produces the recognition sequence for a restriction enzyme, HindIII. Samples without the SNP (not carrying the disease) do not contain the recognition sequence, and are not cut. This allows the digested DNA fragments to be analysed by being separated with the use of gel electrophoresis, enabling the possibility of identifying whether a patient has sickle cell anaeminia. Before we can visualise the DNA we need to amplify the DNA. In order to amplify the DNA we are going to use a technique called Polymerase chain reaction (PCR). With the invention of the PCR technique, DNA profiling took huge strides forward in both discriminating power and the ability to recover information from very small (or degraded) starting samples. PCR greatly amplifies the amounts of a specific region of DNA, using primers and DNA polymerase. The PCR method is readily adaptable for analysing STR loci. The PCR method relies on cycles of repeatED heating and cooling of the reaction for the denaturing of DNA and enzymatic replication of the DNA primers containing complimentary DNA to the target region, with the DNA polymerase synthesising the reaction. The DNA polymerase used is normally Taq polymerase as it is very thermostable. As the PCR progresses, the DNA generated ... ... middle of paper ... ...ow any bands, which shows there has not been any contamination. These results imply that the experiment has been a success as no problems were found. All five samples were used to increase the reliability of the experiment. The PCR without DNA is used as a negative control, no band was expected which ensures that there is no effect when there should be no effect. The PCRs with DNA which are not cleaved are to used compare the cut and uncut DNA. Also, it ensures that there is an effect when there should be an effect. An improvement to the experiment could be including a digest containing fragments of known sizes to be used as a guide to confirm that the cleaved DNA is of the expected size References http://peds.oxfordjournals.org/content/13/4/283.full http://ghr.nlm.nih.gov/handbook/genomicresearch/snp http://www.ndsu.edu/pubweb/~mcclean/plsc431/students/firas.htm

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