Question

I need help to construct this lab report ... I need a well explained introduction and basics methods.

******* PLEASE DO NOT RESPOND IF YOU DONT HAVE THE INFORMATION I NEED***********

INTRODUCTION

Introduce the overall idea behind our lab experiments using buccal cells

1. Real time PCR, using SYBR Green dye

2. Genotype DNA SNP (warrior-worrier gene)

3. Genotype DNA for SNP (ACTN3 gene)

METHODS

Isolation and dilution of DNA: summarize the method used to extract and dilute your gDNA.

1. SYBR Green-based qPCR reaction: provide theoretical background on these

techniques- and summarize the set up of master mix so that a student who never done it can

understand what you have done and how to do it.

2. Taqman-based qPCR reactions: provide theoretical background on these

techniques- and summarize the set up of master mix so that a student who never done it can

understand what you have done and how to do it. Also highlight what the major change between Taqman and SYBR

Answer 1

The struggle of obtaining blood samples in order to acquire high-quality genomic DNA has directed different scientists to establish alternative methods such as collection of buccal cells in a preliminary study which could be subsequently adapted on a large scale. Moreover, clinical validation studies revealed the patient-matched DNA diagnostic results are comparable between buccal cells and whole blood samples.

Following the isolation of DNA from buccal samples, the concentration should be determined using NanoDrop and a concentration of 5 ng/µl should be obtained for each sample.

For each reaction, SYBR Green PCR Master Mix (20 µl reaction mix) should include 1.4 µl DNA (5ng/µl), 10 µl of 2X SYBR, 0.2 µl primer, and 8.4ul of water. Samples and controls were grouped together, randomly intermixed, marked, and loaded in triplicate on the well plates. To generate standard curve, each plate comprised a negative (water) control and serial dilutions of a control DNA sample.

The worrior (Monoamine Oxidase A promoter- MAOA) gene, contains a 30 bp VNTR in the 5' regulatory region of the gene, which affects the gene expression. The product of MAOA gene is located in the nigrostriatial pathway of the brain and is mainly responsible for the degradation of dopamine, serotonin, and norepinephrine. For this polymorphism a genotype by environment interaction has been stated.

There is increasing evidence for strong influence of ACTN3 polymorphism in elite athletic performance affecting muscle fiber composition. The ACTN3 gene produces a molecular protein named alpha-actinin-3 which is only found in fast twitch muscle fibers.

For identification of a gene both SYBR and Taqman is used. The issue with SYBR assays includes generation of PCR products varying in melting temperature. Primer dimers or off-targets with the same melting temperature could indicate a wrong result.

With Taqman this issue can be fixed. The probe sequences for this assay has to fit the target gene, therefore primer dimers or off-target amplification is not noticeable in the results providing additional safety.

For quantification Taqman is the only option to discriminate the target genes.