Polymerase chain reaction (PCR) is an artificial in vivo (outside of the cell) process simulating DNA replication of a selected segment, allowing the selection and rapid amplification of a DNA fragment. The PCR machine is a thermal cycler which controls the temperature inside the container to a very accurate scale, facilitating PCR.
Reaction mixture is the aqueous solution containing all the necessary ingredients (eg. DNA, dNTPs, Buffer, Primer and DNA Polymerase) , in performing PCR. It will be directly heated and cooled in the PCR thermal cycler, which allows for the denaturation (separation) of DNA strands, annealing of primer and extension of dNTPs.
(3’ to 5’)
4. Repeating the cycle
The lengths of primers are usually 18-22 bp. The primers bind to the template by complementary base pairing. The new strand of DNA is produced from 3’ to 5’ of the template strand. Therefore,
Design of Primer 1: forward, complimentary to start of gene
Design of Primer 2: reverse, end of gene
If you wish to know more about how to maximizing the yield of PCR by optimizing the primers, you can read
Alternatively, you may choose to use PCR primer designing tools to aid your design. This is a recommendation of such tool,
Sample for gel electrophoresis or sequencing
Design the primer to bind to a specific mutated sequence. Then run gel to check if the DNA has that sequence.
For example, anemia involves a mutation in the haemoglobin gene. PCR primers are designed to bind to the mutated sequence. Therefore, after amplification, mutated gene is shorter than the normal gene. By comparing the mutated and normal gene by gel electrophoresis, the mutated DNA is further from the well than the normal gene.
Detection of mutation in a gene by cleavage location
Amplification of DNA increases the successful rate and yield of restriction ligation.