Biomedical techniques have been themainstay in the process of the development of diagnostics, therapeutics andtreatment of diseases as well as general research and deciphering the differentmysteries of the cellular systems in general to various dimensions of inquiryand investigation. The advent oftechniques in molecular biology, specifically the analyses of the structure ofDNA, as well as RNA, and other such associated methods, has allowed theidentification of various parameters in testing. It is applicable in thescreening of mutations, chromosome anomalies, differences or alterations inprotein functioning, etc., and overall has been a great boon for the prospectsof the biomedical industry, forensics, genetics, molecular biological research,evolution and others. In the laboratory course, the initialstage involved the isolation of DNA that was followed by the use of thepolymerase chain reaction (PCR) for DNA amplification and gel electrophoresisfor identification of specific proteins as well as their fractions. Thistechnology is essential for use in forensics and crime scene investigation aswell as DNA identification of the suspect and even parental identificationissues.
Moreover, it is a valuable technique used in the study of genetics, DNAanalysis of different organisms, protein identification and detection ofdiseases, many in their early stages, screening populations for particularsusceptibility, etc. PCR empowers the use of techniques to multiply the amountof DNA and hence determine identification, as well as being essential inmolecular genetic analysis. In the first lab, the purification of the productusing TaqMan probe based systems, High Resolution Melt (HRM) analysis,allele-specific PCR, restriction fragment length polymorphism (RFLP) analysisand sanger sequencing were carried out providing a solid foundation as well asthe base for the use of the technology in future applications.
In the secondlab, analysis of RNA was performed; hence, the cell culture and its methods andthe process of isolation of RNA from the cells and preparation of cDNAsynthesis were performed along with the qPCR analysis of the RNA by gelelectrophoresis. Finally, the process of protein analysis was undertaken, andcomparative protein analysis between different cell lines was performedfollowed by SDS-PAGE and western blotting. These technologies are mostextraordinarily sophisticated, and exposure to these techniques has been acomplete package for the analysis of the cellular machinery at the moleculargenetic level. This could have profound applications in the detection ofdiseases, protein malformations, mutation studies, genetics and molecularbiology, etc. Evaluation of five different methods for detection SNP (rs4680) Abstract Catecholaminesare degraded by a number of enzymes, including catechol-O-methyltransferase(COMT).
COMT, which in humans is encoded by the COMT gene, is present in twoisoforms: a soluble short form (S-COMT) and a long, membrane-bound form(MB-COMT). COMT’s mechanism of degrading catecholamines is to transfer to it amethyl group from S-adenosyl methionine (SAM). COMT has numerous substrates,including all compounds bearing catechol structures, such as catecholestrogen,and flavonoids with catechol moieties.
The aim of this study is to evaluate theeffectiveness of allele-specific PCR, high resolution melt (HRM), restrictionfragment length polymorphism (RFLP), Sanger sequencing and TaqMan probe-basedmethods in detecting the rs4680 SNP in COMT. Introduction Singlenuclear polymorphism (SNP) is a term generally used to describe the variationof a single base pair in a gene. An example of SNP is the gene encodingcatechol-O-methyltransferase (COMT), an enzyme that breaks downneurotransmitters in the brain. The SNP, rs4680, is a single nucleotidevariation, which is located in the COMT gene on chromosome 22, position19963748 (figure 1.1). Polymorphism rs4680 (Val158Met) arises in COMT from asingle base pair substitution of guanine (G) for adenine (A), resulting in thecoding of methionine amino acid at the 158th position of the genetic code inplace of valine. (Snpedia.
com, 2018). This SNP alters the structure of COMT,which results in the allele A (Met) having reducedfunctional enzyme activity, compared to the COMT activity of the G allele. COMTfunctions by transferring the methyl group from an adenosyl methionine tocatecholamines that are found predominantly in the nervous system. COMT istherefore a vital enzyme in the degradative pathways of the body. It functionsin the metabolism of catechol drugs used in the treatment of diseases such asParkinson’s disease (Snpedia.
com, 2018). It exists in the body in two forms:the membrane-bound form and soluble form found in circulation. As presented in figure1.2 and 1.3, bioinformatics show A and G specific alleles.