Polymerase chain reaction

From Ganfyd

Developed by Kary Mullis who was awarded the 1993 Nobel prize for Chemistry for the idea. Mullis describes the origin of the technique both in his Nobel lecture [1] and a 1990 Scientific American article [2]

A biomolecular technique developed in the 1980s^[3] that replicates specific stretches of DNA to create multiple copies. The main advantage to the method is that it creates a much larger volume of DNA than in the original sample. An example might be a sample containing 100,000 cells, which will only contain 100,000 DNA molecules, rendering most tests impossible.

Applications

• Microbiological diagnosis, e.g. identifying the presence of organisms, e.g. Chlamydia, TB, bacteria, HIV, measles, meningococcus... Very small amounts of microbial nucleic acid can be detected using this method, which can allow confirmation of diagnosis (and often some information about e.g. the serotype or strain) when conventional methods, such as bacterial or viral culture, cannot be used.
• DNA analysis, e.g. looking for mutations or specific sequences
• Forensic
• Paternity testing

Methodology

Requirements

Template DNA

Usually derived from cells, from which DNA can be extracted. Blood is another common source. Any source can act as a source of uncontaminated genomic sample, but a degree of contamination can be overcome by adjusting the reaction conditions to increase specificity, e.g. the use of longer primers and higher annealing temperatures.

Alternatively, nested PCR, which involve 2 sequential PCR reactions using different primers allows further specificity. As an example, it is possible to identify mutated human DNA sequences derived from colonic tumour cells in stool.^[4]

DNA polymerase 

The polymerase used was originally derived from Thermophilus aquaticus, a bacterium that lives at high temperatures (Taq polymerase). Other similar bacteria include Pyrococcus furiosus (Pfu polymerase). Most polymerase today is produced with recombinant technology.

Nucleic acid primers 

Short oligonucleotide sequences, usually about 16-25 base pairs long.

Individual nucleotides 

For incorporation into the DNA chains

Heat

to provide energy for the reaction

Co-factors

Magnesium, and in difficult stretches, dimethyl sulfoxide.

These heat-stable polymerases allow DNA amplification by alternately unzipping the double helix with heat and new chains forming on the exposed templates without the enzyme denaturing. The process requires a pair of primers. These are custom-made oligonucleotide sequences that flank the stretch of DNA of interest.

The process starts with initial denaturing period (e.g. 5 minutes), usually at a temperature of around 95°C to unzip the DNA present. This is followed by an annealing step. The temperature is lowered to allow the primers to bind to the complementary sequence on the DNA template. The exact temperature depends on the make-up of the primers. Once the primers have had time to anneal, the next stage is an elongation stage at about 72°C, the optimum temperature for the Taq polymerase to function. The cycle of annealing, elongation and denaturing is then repeated 25-35 times (although during the cycling stage, the denaturing stage is shorter, e.g. 30 seconds at 95°C). As the reaction is (mostly) exponential, 25-35 cycles produces significant amplification. A final elongation step is required to complete any incomplete products.

see http://www.abpischools.org.uk/resources/poster-series/pcr/pcr.pdf

References