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Real Time Polymerase Chain Reaction (PCR) Instruments Maintenance Management, Support, and Repair

How it works

Real Time Polymerase Chain Reaction (Real Time-PCR) is a technique based on the polymerase chain reaction to amplify and simultaneously quantify a targeted DNA molecule. PCR allows exponential amplification of short DNA sequences (usually 100 to 600 bases) within a longer double stranded DNA molecule. This entails the use of a pair of primers, each about 20 nucleotides in length that are complementary to a defined sequence on each of the two strands of the DNA. The double stranded DNA is melted by raising the temperature to above 90oC then cooling to allow the primers to bind to the denatured DNA. The primers are extended by a thermo-stable DNA polymerase (Taq polymerase) so that a copy is made of the designated sequence. After making this copy, the same primers can be used again, not only to make another copy of the input DNA strand but also of the short copy made in the first round of synthesis. This leads to logarithmic amplification. In real-time PCR fluorescent molecules are attached to the amplified cDNA. Real Time PCR machines are able to measure this fluorescence. Thus the amplified DNA is quantified as it accumulates in the reaction in real time after each reaction cycle. As the amounted of copied DNA increases the amount of fluorescence increases and can be displayed on a graph.

Two common methods of quantification are the use of fluorescent dyes that intercalate with double stranded DNA and modified DNA oligonucleotide probes that fluoresce when hybridized with a complimentary DNA. The real-time machines not only monitor DNA synthesis during the PCR, it can also be used to determine the melting point of the product at the end of the amplification reactions. The melting temperature of a DNA can be used to determine the authenticity of the product because the double helix depends on its base composition (and its length if it is very short). All PCR products for a particular primer pair should have the same melting temperature - unless there is contamination, mispriming1, primer-dimer2 artifacts, or some other problem. These attributes of Real Time PCR allow rapid identification of biological agents present in minute amounts.

KD Analytical supports PCR Instruments

KD Analytical offers a suite of services for PCR instruments (as well many other analytical instruments) that can save you money and time and help you to guarantee the readiness of your equipment and team.

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