Biomedical valuable technique used in the study of

Biomedical techniques have been the
mainstay in the process of the development of diagnostics, therapeutics and
treatment of diseases as well as general research and deciphering the different
mysteries of the cellular systems in general to various dimensions of inquiry
and investigation.  The advent of
techniques in molecular biology, specifically the analyses of the structure of
DNA, as well as RNA, and other such associated methods, has allowed the
identification of various parameters in testing. It is applicable in the
screening of mutations, chromosome anomalies, differences or alterations in
protein functioning, etc., and overall has been a great boon for the prospects
of the biomedical industry, forensics, genetics, molecular biological research,
evolution and others.                                                                                                  

In the laboratory course, the initial
stage involved the isolation of DNA that was followed by the use of the
polymerase chain reaction (PCR) for DNA amplification and gel electrophoresis
for identification of specific proteins as well as their fractions. This
technology is essential for use in forensics and crime scene investigation as
well as DNA identification of the suspect and even parental identification
issues. Moreover, it is a valuable technique used in the study of genetics, DNA
analysis of different organisms, protein identification and detection of
diseases, many in their early stages, screening populations for particular
susceptibility, etc. PCR empowers the use of techniques to multiply the amount
of DNA and hence determine identification, as well as being essential in
molecular genetic analysis. In the first lab, the purification of the product
using TaqMan probe based systems, High Resolution Melt (HRM) analysis,
allele-specific PCR, restriction fragment length polymorphism (RFLP) analysis
and sanger sequencing were carried out providing a solid foundation as well as
the base for the use of the technology in future applications. In the second
lab, analysis of RNA was performed; hence, the cell culture and its methods and
the process of isolation of RNA from the cells and preparation of cDNA
synthesis were performed along with the qPCR analysis of the RNA by gel
electrophoresis. Finally, the process of protein analysis was undertaken, and
comparative protein analysis between different cell lines was performed
followed by SDS-PAGE and western blotting.

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These technologies are most
extraordinarily sophisticated, and exposure to these techniques has been a
complete package for the analysis of the cellular machinery at the molecular
genetic level. This could have profound applications in the detection of
diseases, protein malformations, mutation studies, genetics and molecular
biology, etc.

Evaluation
of five different methods for detection SNP (rs4680)

Abstract

 

Catecholamines
are degraded by a number of enzymes, including catechol-O-methyltransferase
(COMT). COMT, which in humans is encoded by the COMT gene, is present in two
isoforms: 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 a
methyl 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 the
effectiveness of allele-specific PCR, high resolution melt (HRM), restriction
fragment length polymorphism (RFLP), Sanger sequencing and TaqMan probe-based
methods in detecting the rs4680 SNP in COMT.

 

 Introduction

 

Single
nuclear polymorphism (SNP) is a term generally used to describe the variation
of a single base pair in a gene. An example of SNP is the gene encoding
catechol-O-methyltransferase (COMT), an enzyme that breaks down
neurotransmitters in the brain. The SNP, rs4680, is a single nucleotide
variation, which is located in the COMT gene on chromosome 22, position
19963748 (figure 1.1). Polymorphism rs4680 (Val158Met) arises in COMT from a
single base pair substitution of guanine (G) for adenine (A), resulting in the
coding of methionine amino acid at the 158th position of the genetic code in
place of valine. (Snpedia.com, 2018). This SNP alters the structure of COMT,
which results in the allele A (Met) having reduced
functional enzyme activity, compared to the COMT activity of the G allele. COMT
functions by transferring the methyl group from an adenosyl methionine to
catecholamines that are found predominantly in the nervous system. COMT is
therefore a vital enzyme in the degradative pathways of the body. It functions
in the metabolism of catechol drugs used in the treatment of diseases such as
Parkinson’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 figure
1.2 and 1.3, bioinformatics show A and G specific alleles.