Hi I am working on my biochemistry lab report called Polymerase Chain Reaction a
ID: 271548 • Letter: H
Question
Hi I am working on my biochemistry lab report called Polymerase Chain Reaction and Gel Electrophoresis. I have no clue what I should write for my discussion/conclusion. Plus I dont really get this lab. It will be really helpful if you could help me out with my discussion part. I have attached the whole lab manual and the results obtained for your referrence. Please help!
Practical 5 Individuals can be categorized into two predicted phenotypes based on the observed CIP2CI9 genotypes, as shownin the table as below (Gurbel et al 2010) POLYMERASE CHAIN REACTION AND GEL ELECTROPHORESIS Predieted Phenotype Poor metabolizar PM) Observed CIP2C19 Genotypes ?1/*3, *3/*3 metabolizer INTRODUCTION Polymerase Chain Reaction (PCR) is a method used to synthesize and amplify specific region of template sequencin aDNA strand Thenewly synthesizad and amplified DNA sequence is complementary to the specific region in the original template strand. At the end of a PCR reaction, billons copies of the specific DNA templates (also known as amplicons) will be accumulated (PCRnd) CIP2CI9 *3 allele has apoint mutation of 636 GA in exon 4, leading to premature stop codon and a defected BamHI restriction site. Therefore, homozygous mutant alleles showad single band undigasted), homozygous wild type showed two bands (digasted) and heterozygous alleles showad three bands (both digestad and undigestad) Teiri et al. 1996). The homozygous mutant of CIP2CI9 showed single band of 27l bp. The homozygous wild type of *3 (175 bp and 96 bp) showed two bands. The heterozygous CIP2C19 *3 (271 bp, 175 bp and 96 bp) displayed three bands (Teiri et al. 1996). Crucial components required for PCR include: (ii) DNA polymerase Enzyme that synthesizes new DNA strand that is (iii) Forward and Reverse Primers - A short, single stranded DNA that is iv) Nucleotides Deoxynucleotide triphosphates (dNTPs) -Buildingblocks for DNA template - DNA sample with desired sequence complementary to theDNA template complementary to the targeted sequence DNA polymerase will synthesiza Chemicals and Materials: new DNA from the primers nds MasterMix (Consisting of dNTPS, DNA Taq polymerase, DNA Taq polymerase buffer CYP2C19*3 forward and reverae primers, MgC) DNA Loading Buffer DNA ladder 1xTAE buffer Sterile deionisad water s-dH O) Male andFemale DNA samples Microcantrifuge tubes PCR tubas Agarose gal elactrophoresis systam Midori green staining solution 1.0%Agarose gel new DNA strands which consist ofsingle units of A, T, G and C bases. Gel electrophoresis is a chromatographic method that separates and purifies proteins and nucleic acids that differ in molecular sizes, ionic charges or molecular conformation (Principles of Gel Electrophoresis 2000) In order to check a PCR reaction, gel electrophoresis is often used. When the DNA template of interest is the only sequenca that is being amplified after a PCR reaction, a single band should be obtained in the gel Contrarily, ifmore than one template sequence is beingamplified; rultiple bands wil be obseved on the gel For this practical, you will leam the technique of preparing samples for PCR reaction operate a PCR machine, and check the amplified PCR product with gel electrophoresis. You will be using PCR technique to amplify CIP2Ci9 3 gene from male and female DNA samples provided REMINDER: Sprav 70 % ethanol onto your glove. This is to sarnrite your glove to noid cross In humans, CIP2CI9 protein is part of the cytochrome P450 (CYP) functional system which is encoded by the CIP2Ci9 gene and it regulates the metabolism of certain groups of drues. Clopidogrel is a type of inactive antiplatelet prodrug that requires hepatic bioactivation by CYP enzymes, which also includes the CIP2CI9 protein The enzymatic activity of CIP2CI9 is highly dependent on the allele present ofwhich its activity can either be nomal, reduced or increased (Shah 2011) To date, 6 different CIP2Ci9 alleles have been identified. The *3 (Star 3) allele is one ofthe alleles that result in complete loss of enzymatic activity. Hence, during the course of Clopidogrel treatment, carriers of *3 allele will experienced reduced madical effects in tems ofClopidogrel-induced platel:t inhibition (Shah 2011) Method Prepare THREE differant samples for PCR reaction in accordance to the racipe as follows and label them accordingly; 1. 27 28Explanation / Answer
Using gel electrophoresis to to visualise the results of PCR . It is the technique innwhich fragments o DNA are oulled through gel matrix by an electric current