(b) Polymerase chain reaction (PCR) amplification of DNA using complementary primers for specific target sequences. DNA heated to separate strands then cooled for primer binding. Heat tolerant DNA polymerase then replicates the region of DNA. Repeated cycles of heating and cooling amplify this region of DNA. Practical applications of PCR.
Polymerase chain reaction, or PCR, is a laboratory technique used to make multiple copies of a segment of DNA. PCR is very precise and can be used to amplify, or copy, a specific DNA target from a mixture of DNA molecules. The following steps are used. Setting up the tubes:
1. two short DNA sequences called primers are designed to bind to the start and end of the DNA target.
2. to perform PCR, the DNA template that contains the target is added to a tube that contains primers, free nucleotides, and an enzyme called DNA polymerase, and the mixture is placed in a PCR machine.
3. the PCR machine increases and decreases the temperature of the sample in automatic, programmed steps.
Interactive animation of PCR
The temperature changes which form the steps in the PCR process are necessary as a result of the the way in which DNA polymerase works.
1. 920-96o–C – DNA polymerase only works on single stranded template DNA, so the relatively high temperature is necessary to break the hydrogen bonds and create single stranded template DNA. (this is also why we use Taq polymerase a high temperature version of DNA polymerase).
2. Cooling to aorund 55o-C – the primers will not bind to the template strand at high temperatures (hydrogen bonds break at these temperatures), so cooling is required to allow the primers to bind (anneal).
3. Heating again to around 72o-C – this allows the Taq polymerase to work efficiently to produce replicate the DNA strand of the target DNA.
Carrying out a cycle:
a) Initially, the mixture is heated to between 92o–97oC to denature, or separate, the double-stranded DNA template into single strands.
b) The mixture is then cooled so that the primers anneal, or bind, to the DNA template.
c) At this point, the DNA polymerase begins to synthesize new strands of DNA starting from the primers.
Following synthesis and at the end of the first cycle, each double-stranded DNA molecule consists of one new and one old DNA strand. PCR then continues with additional cycles that repeat the aforementioned steps. Every cycle doubles the number of copies of target DNA present, because the newly synthesised DNA segments serve as templates in later cycles, giving exponential amplification up to millions of times.
Primers & specificty of PCR
The key to the specificity of PCR is the primers. The primers are designed to locate a DNA sequence on either side of the target gene. The primer is exactly complementary to a short (20-30 bases) sequence of DNA on either side of the target and so will bind only to that sequence. In this way the primer can be considered to find the “needle in the haystack”. PCR then produces millions or billions of copies of this single sequence – producing “haystacks from needles”.
A short primer will be less specific. The chance of any base appearing at any position in the genome is one in 4 (4 bases). Therefore if your primer is only 2 bases long there is a 1 in 16 chance of encountering that random 2 base sequence in the genome. Given that the genome is 3 billion or so bases long – you will likely encounter a 2 base sequence very often – so that primer will not provide specificity. A 20 base primer gives a 1 in 1,099,511,627,776, that’s a 1 in a trillion chance of encountering the same sequence randomly in the genome.
Applications of PCR
PCR is simply a technique to amplify DNA, but if appropriate regions of DNA are amplified, PCR becomes a very powerful application in a variety of contexts. It is particularly useful if there are only small amounts of DNA available.
Infection diagnosis : PCR can be used to amplify specific parts of a pathogen’s genome to help identify the cause of an infectious disease
Paternity/ Family tree : PCR is able to be used in providing confirmation of family relationships. It relies on the identification of differences in the repeated sequences present in geneomes (the so-called genetic fingerprint). These are inherited from both parents.
Forensics : PCR enables the analysis of miniscule amounts of DNA and consequent identification of the person responsible for leaving that DNA at crime scenes. It has also been used to exonorate serving prisoners and can be important in reversing miscarriages of justice.
Anthropology : PCR can be used to analyse the small amounts of DNA found in historic artefacts helping to identify relationships between individuals and sometimes uniquely identify individuals who died hundreds or thousands of years ago.
Phyolgeny : PCR can help in the analysis evolutionary relationships between different species.