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Molecular Biology: PCR Techniques Author: Prof Estelle Venter Licensed under a Creative Commons Attribution license. Table of Contents INTRODUCTION 2 The principle of PCR 2 MATERIALS AND METHODS 5 Components needed 5 The different steps in PCR 6 CONTAMINATION 12 OTHER PCR’s 13 Reverse transcription PCR 13 Random amplification of polymorphic DNA 14 Multiplex PCR 14 Nested PCR 14 Touchdown PCR 14 Hot Start PCR 15 Real-time PCR 15 (DIAGNOSTIC) APPLICATION OF PCR 15 THECHNIQUES USED IN PCR DIAGNOSTICS 17 FAQs 18 REFERENCES 20 INTRODUCTION The polymerase chain reaction (PCR) is a simple method for producing unlimited copies of a specific DNA sequence in a test tube which allows a “target” DNA sequence to be selectively amplified several million-fold in just a few hours. The PCR achieves amplification of a predetermined fragment of DNA, (the target; which can e.g. be from 100 – 1000 bp long) with the apparent disadvantage that the sequences flanking the target region must be known, the latter precludes the use of PCR from analysis of DNA regions that have not previously been studied by standard methods. The principle of PCR The PCR is used to amplify a sequence of DNA using a pair of oligonucleotide primers each complementary to one end of the DNA target sequence. High temperatures are used to separate the DNA molecules into single strands, and the synthetic sequences of ss DNA (18-30 nucleotides) serve as primers. One primer is complementary to the one DNA strand at the beginning of the target region; a second primer is complementary to a sequence on the opposite DNA strand at the end of the target region. 5’-TTAACGGGGCCCTTTAAA..target sequence..TTTAAACCCGGGTTT-3’ Positive DNA strand5’-TTAACGGGGCCCTTTAAA-3’Primer 1and:3’-AAATTTGGGCCCAAA-5’ Primer 23’-AATTGCCCCGGGAAATTT..target sequence..AAATTTGGGCCCAAA-5’ Negative DNA strand Location of PCR primers These are extended towards each other by a thermostable DNA polymerase in a reaction cycle of three steps: denatu