Beginner’s Guide to RealTime PCR （实时PCR初学者的指南）.pdf

Beginner’s Guide to Real-Time PCR Real-time PCR 02 basic principles PCR or the Polymerase Chain Reaction has become the During a PCR, changes in temperature are used to control cornerstone of modern molecular biology the world over. the activity of the polymerase and the binding of primers. Real-time PCR is an advanced form of the Polymerase Chain Reaction that maximizes the potential of the To begin the reaction the temperature is raised to 95°C. technique. At this temperature all double stranded DNA is “melted” in to single strands: To understand real time PCR it is easier to begin with the principles of a basic 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. DNA analysis requires amplification in order for there to be enough DNA to give a detectable signal for quantification. If you amplify both samples at the same rate, you can calculate which sample had the highest copy number of the target of The temperature is then lowered to ~60°C. This allows interest to begin with. the primers to bind to your gene of interest: It is a thermostable polymerase enzyme tha