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PCR Procedures[ Qualitative PCR Analysis | Quantative PCR Analysis | Detection Limit & Quantification Limit ] PCR is a method that selectively generates copies of a defined section of an original DNA molecule that serves as a template. The targeted DNA region can thereby be reproduced from small numbers of molecules to detectable levels. This in vitro reaction that multiplies a specific DNA region is performed by a 'DNA copying' enzyme (DNA-polymerase). It requires the presence of a template for the DNA sequence in question. To construct the copies of the targeted DNA, the process uses nucleotide "building blocks" supplied by the reaction mixture. Moreover, the DNA-polymerase requires starting points for its activity. These are provided and exactly defined by the addition of the so-called primers, short pieces of synthesized DNA that are complementary to the regions surrounding the targeted DNA sequence. Since the reaction mixture is heated multiple times (see below), DNA-polymerases used for PCR have to be resistant to high temperatures (e.g. Taq-polymerase from the thermophilic microorganism Thermus aquaticus that is found naturally in thermal springs). To analyze an agricultural or food product by PCR testing, first an aqueous solution of the DNA present in the sample must be obtained. Therefore the DNA is extracted from the sample and purified to a degree that makes it suitable to serve as a template in the subsequent PCR reaction. The sample DNA is combined with the other components of the PCR reaction and placed into a thermocycler. A thermocycler is a device that can be programmed to alternately raise and lower the reaction temperature to specific levels for defined time periods. Initially, the double-stranded DNA molecule is separated into two complementary, single strands by heating. Afterwards, at a lower temperature, the primers recognize their specific target DNA sequences on each single strand of the DNA. Only if the sample DNA contains the DNA sequences in question, targeted by the primers, will the primers anneal to the single-stranded templates and thereby mark the starting points for the synthesis of the complementary strands. In the double-helix of the DNA molecule, the two nucleotide strands are paired by complementary interactions between the bases adenine (A), thymine (T), cytosine (C) and guanine (G); these base pairs form the rungs of the ladder-like DNA molecule. Bonding only occurs between adenine and thymine or between cytosine and guanine. A new DNA strand is built from each template DNA strand as the complementary bases are progressively added (A&T, C&G). At the end of the first PCR cycle, two complete double-stranded DNA molecules result from each original double-stranded DNA molecule present at the beginning of the PCR. In each successive cycle, these newly formed double-stranded DNA molecules serve as templates as well, and each will yield two copies. As the number of molecules theoretically doubles with each cycle, multiple repetitions of the PCR cycle will amplify the targeted DNA region to detectable amounts. The products of the PCR reaction can then be visualized by agarose gel electrophoresis. Since the increase of target DNA molecules during the PCR process theoretically is exponential until the supply of the reaction's components is exhausted, the number of PCR cycles can be crucial for the sensitivity of the analysis. Especially if only very few target DNA molecules are present in the beginning of the reaction, ten cycles more or less can easily be critical to obtain a detectable amount of PCR products. Typically, GeneScan USA and all other GeneScan laboratories use 50 PCR cycles. » Download the Sensitivity
of a PCR Analysis Word Document
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