Amplification Of Exons 2 And 4 - 1586 Words

Amplification Of Exons 2 and 4 To Detect Mutations In The HFE Gene Of Human DNA That Leads To Iron Overload Causing Hemochromatosis Introduction The HFE gene, that causes the disease officially known as hemochromatosis, is found on the short arm of chromosome six (Dostalikova-Cimburova et al., 2012). This HFE gene codes for a protein that is found on the surface of liver, intestinal, and immune cells (D’Alessio et al., 2012). The HFE protein interacts with many other proteins to cooperatively regulate the amount of iron present in the body. The HFE protein regulates a very important protein called hepcidin. Hepcidin is the main trans membrane protein that uses hormones to regulate iron in the body (Van Dijk et al., 2008). When there†¦show more content†¦The second major mutation of the HFE gene is the G845A nucleotide change in exon 4, which alters the availability of the RsaI restriction sites (Barton et al., 2005). This is because a substitution is made for the amino acid sequence C282Y in the protein (Trifa et al., 2012). The purpose of the laboratory experimentation is to screen human genomic DNA for mutations in the HFE gene, which are linked to hereditary hemochromatosis (Bates et al., 2008). This is important because by amplifying the exons 2 and 4 within the HFE gene the single nucleotide mutations will be detected and analyzed to better understand the cause of hemochromatosis. The mutations of main focus as mentioned previously are the C187G nucleotide change within exon 2 and the G845A nucleotide change within exon 4. These are the main focus because they directly alter the shape and function of restriction sites necessary for regulation of iron in the body. The experimentation was completed using common techniques of PCR amplification, restriction digest, and gel electrophoresis. Materials and Methods An important step in detecting the single nucleotide changes within the HFE gene that causes hemochromatosis is to first use the process of Polymerase Chain Reaction (PCR) amplification to amplify specific parts of exons 2 and 4. In the PCR amplification specific primers were used independently for each exon 2 and