PCR is a technique used to make many copies of a small section of DNA.
WHY would you want to do this?
DNA is extremely small and impossible to see directly. This makes it very difficult for scientists to analyse. PCR allows a scientist to make millions of copies of a small section of DNA. This makes it easier for the DNA to be sequenced or to work out if a mutation has occurred in that part of the DNA.PCR is a bit like following a cooking recipe. First you must measure out all the ingredients that then go through a series of cycles to make the many copies of the DNA you want amplified.
What goes into the PCR mix?
1. The DNA sample that you wish to amplify: This can be extracted from any cell that contains DNA, which includes all human cells (except red blood cells).
2. Two primers: These are short, single stranded pieces of DNA that are complementary to regions of DNA on either side of the section you wish to amplify. These are synthesised in a laboratory to have the exact sequence required, and are added to the PCR reaction in abundance.
3. Thermostable DNA polymerase: This is an enzyme that replicates DNA. It is found in all cells.The DNA polymerase used in PCR needs to be resistant to high temperatures (thermostable) so that the high temperatures in the thermocycler don’t destroy it. (One of the most common DNA polymerases used in PCR comes from the bacterium (Thermus aquaticus) which lives in thermal springs.)
4. DNA nucleotides: These are the building blocks used to make the DNA copies. Nucleotides of all four types (bases) are added – A, T, C and G.
5. Buffer solution: This is a solution that contains salts and other chemicals that help the polymerase to function.
So, how is PCR done? A number of steps are involved:All the ingredients are combined in a small tube (called an eppendorf tube), and placed in a thermocycler machine (Fig 2), that regulates the temperature in 3
distinct steps:
1. Denaturation: The PCR mix is heated to ~95°C.This high temperature weakens the hydrogen bonds between the bases in the double-stranded DNA sample, causing the two strands to separate.
2. Attachment of primers: The mixture is cooled to between 50 and 55°C. As this happens, primers attach (anneal) themselves to the newly separated DNA strands on either side of the section to be amplified.
3. Extension: The PCR mix is now heated to 72°C. This allows the DNA polymerase to bring free nucleotides into the sequence, forming a complementary copy of each of the original DNA strands. The primers are used as starting points for the DNA polymerase to start adding to.
This cycle of three steps is repeated about 30 times, with each cycle resulting in a doubling of the amount of DNA between and including the two primer
sites.
Figure 2: A PCR thermocycler
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