《Beginners guide to real-time PCR》.pdf

Beginner’s guide to Real-time PCR Beginner’s guide to Real-time PCR PCR or the Polymerase Chain Reaction has become the cornerstone of modern molecular biology the world over. Real-time PCR is a bespoke form of the Polymerase Chain Reaction that maximizes the potential of the technique. To understand Real Time PCR it’s easier to begin with the principles of a basic PCR Principles of PCR PCR is a technique for amplifying DNA. There are 2 reasons why you may want to amplify DNA. Firstly you may want to simply create multiple copies of a rare piece of DNA. For example a forensic scientist may want to amplify a tiny piece of DNA from a crime scene. More commonly however you may wish to compare 2 different samples of DNA to see which is the more abundant. Because DNA is microscopic you cannot see which sample contains the most DNA. However, if you amplify both samples at the same rate, you can calculate which sample was the biggest to begin with by establishing which is the biggest after amplification. It is a Polymerase enzyme that drives a PCR. A polymerase will synthesize a complementary sequence of bases to any single strand of DNA providing it has a double stranded starting point. This is very useful because you can choose which gene you wish the polymerase to amplify in a mixed DNA sample by adding small pieces of DNA complimentary to your gene of interest. These small pieces of DNA are known as primers because they prime the DNA sample ready for the polymerase to bind and begin copying the gene of interest. During a PCR, changes in temperature are used to control the activity of the polymerase and the binding of primers. To begin the reaction the temperature is raised to 95°C. At this temperature all double stranded DNA is “melted” in to s