PCR Thermal Cycler - Basics
| What does “PCR” mean? | Polymerase Chain Reaction / Reactor / Thermal cycler | ||||
| What does it do? | PCR (Polymerase Chain Reaction) is the method used to produce multiple copies of the original DNA. It is a series of controlled heating and cooling steps performed in a heating block (Polymerase Chain Reactor otherwise known a s a thermal cycler). Concentrating or amplifying the DNA allows it to be measured; in this way detection limits for various diseases have been drastically lowered. | ||||
| Physiology | In most cells the genetic code is built into long molecules called deoxyribonucleic acid (DNA). These large twisted molecules are tightly packaged and form part of the chromosomes in the cell nucleus. The genetic information is copied from DNA to RNA and is eventually translated into the proteins that are needed for life. | ||||
| How it works |
The PCR reaction has 3 steps. The first is heating to 94°C
to denature the double-stranded DNA. Next it is cooled to the
annealing temperature, permitting chemical “primers”
to bind to their specific target. In the third step the temperature
changes to the optimum for an enzyme to manufacture a new strand
of DNA. A typical PCR cycle would be 94°C for 30 seconds followed by 65°C for 30 seconds and an extension step of 72C for 1 minute. These three steps would be repeated for 30 cycles. A PCR cycler is essentially a heating block designed for extremely rapid and accurate temperature change to give maximum yield. The temperature must change at least 2°C per second. The samples and tubes must be as small as possible for best transient results and a very good electronic controller used. Peltier cells are often used to heat and cool the heating block. |
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| Units of measurement | Degrees Celsius °C, Watts | ||||
| Typical values |
Each cycle theoretically doubles the amount of DNA. IE 2, 4, 8, 16, 32, 64,128, 256, 512, 1024 etc. So 10 cycles would give over 1000 times original concentration, whilst 30 cycles would result in an amplification of over 1,000,000,000. In practice the efficiency of the reaction is less than 100%, resulting in smaller yields. | ||||
| Pictures of equipment | |||||
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