End-point PCR

There are many applications for polymerase chain reaction (PCR) in molecular research such as genotyping, mutagenesis, DNA and RNA amplification and introducing restriction sites to cDNA for sub cloning, to name just a few.

Fig. 1 – Agarose gel of various PCR products

Most PCR reactions are performed with similar reagents.

10X PCR Buffer 1X
10 mM dNTP mixture 0.2 mM each
50 mM MgCl2 1.5 mM
Primer mix (5 µM each) 0.33 µM each
Template DNA ~50 ng
DNA Polymerase Units depend on application
Autoclaved, distilled water Not applicable

… and they follow the same basic thermocycling protocol of (25-40) repeating cycles:

  • Denaturation (usually 95 °C)
  • Annealing (varies depending on the primer but usually 50-65 °C)
  • Extension (usually 72 °C)
  • These cycles are followed by a final extension step of 72 °C for 5-10 min

The success of any PCR reaction is directly related to the specificity and thermal properties of the primers used in the reaction.

Primer design principles

When designing primers, consideration should be given to the area of the target gene being amplified (target sequence) and the thermodynamic properties of the primers themselves. All of these properties factor into the efficiency and specificity of target gene amplification. Forward and reverse primer pairs for PCR should bind to unique target sequences between 18-30 bp long that are areas of low secondary structure (GC content < 60%). The primers themselves should follow the guidelines for good primer design (see below), but briefly, they should have low secondary structure (hairpins, loops etc..) and should be “anchored” with a G/C at the 5’ end of the sequence with their melting temperature approximately 5°C higher than their annealing temperature. Primer pairs should have similar thermodynamic properties. There are many good on-line sites for primer design:

Fig 2. – Sample read-out from “Primer BLAST” primer design program

10 steps for good primer design

Aim for 18-30 nucleotides in length

Try to make sure the melting temperatures are between 55° and 65°C and, for a pair, that they are within 5°C of each other

If you have a low Tm primer, find a sequence with more Gs and Cs, or extend the length of the primers slightly

Avoid intra-primer complementary (where there are more than three bases that complement within the primer) or inter-primer complementary (where forward and reverse primers have complementary

Typically, 3-4 nucleotides are added to the 5’ of the restriction site in the primer to allow for efficient cutting

Aim for the GC content to be 40-60%, with a C or G at the 3’ end to promote binding

Avoid regions of secondary structure and have a balanced distribution of GC-rich and AT-rich domains

Try to steer clear of runs of four or more of one base, or dinucleotide repeats

If you are using the primers for cloning, we recommend a minimum level purity by cartridge purification

If you’re using your primers for mutagenesis, have the mismatched bases toward the middle of the primer

**Remember to Blast your primer sequences to confirm recognition of the desired template**