PCR

So it's possible to amplify the exact gene, nothing more.

PCR is an acronym which stands for polymerase chain reaction. The PCR technique is basically a primer extension reaction for amplifying specific nucleic acids in vitro. The use of a thermostable polymerase allows the dissociation of newly formed complimentary DNA and subsequent annealling or hybridization of primers to the target sequence with minimal loss of enzymatic activity.

PCR will allow a short stretch of DNA (usually fewer than 3000 bp) to be amplified to about a million fold so that one can determine its size, nucleotide sequence, etc. The particular stretch of DNA to be amplified, called the target sequence, is identified by a specific pair of DNA primers, oligonucleotides usually about 20 nucleotides in length.

How should I select a set of primers to use for PCR?

Try to keep the primer 50% G-C give or take 15%. If overly G-C rich add a string of As or Ts at 5' end; If overly A-T rich, do the same with Gs and Cs.

Try to avoid Gs and Cs at 3' end of the primers. This may increase the chance of forming primer dimers.

Avoid self-annealing regions within each primer.

Compute Tm as sum of 4 C for G/C and 2 for A/T, then subtract 5 C from this value and that is our annealing temp. Naturally, the annealing temp will be that of the primer with the lower value. Differences of 4-6 C do not seem to affect yield of PCR. Ideally you would like the Tm for each primer to match and be within the 70-75 degrees C range.

A good practice is to check the target DNA sequence if it is known for mispriming areas. A quick check scanning the sequence of vector for approximately 70% and above homolgy regions can help prevent obtaining multiple contaminating bands in your PCR.

Use of a computer program may help eliminate the use of a poorly designed pair of primers.

PCR resources