PCR Limitations and Disadvantages

The High Sensitivity of PCR

Due to the extremely high sensitivity of PCR, contamination from non-template PCR present in the lab environment (from bacteria, viruses, and your own DNA) presents a real problem. Precautions therefore must be taken. Several steps can be taken to prevent and minimize PCR contamination including the use of aerosol barrier tip filters, laminar flow cabinet, and a special preparation area for PCR separate from the DNA isolation area.

Polymerase Errors

DNA polymerase are not perfect by any means, however they are quite good at what they do. Mistakes and errors do happen in PCR by DNA polymerases in the test tube and in the human body. The polymerases used in PCR often lack 3' to 5' exonuclease activity such as Taq polymerase. This enzyme lacks the ability to correct misincorporated nucleotides, an activity which is present in higher eukaryotes.

A polymerase lacking the 3' to 5' exonuclease activity has a higher error rate, around 1 in 10000 bases are misincorporated.

Recombinant polymerases have been generated and other polymerases have been isolated which are available to more accurate PCR and have a variety of applications such as sequencing.

Examples of polymerases with 3' to 5' exonuclease activity include: Vent, which is extracted from Thermococcus litoralis , Pfu which is extracted from Pyrococcus furiosus, and Pwo which is extracted from Pyrococcus woesii .

PCR Polymerase Enzyme Proficiency and PCR Product Size / Length Limitations

DNA polymerase are proficient enough to efficiently amplify DNA products up to a few thousand basepairs (2-5 kb). PCRs of longer products are less efficient due to enzyme activity loss, and inaccuracies introduced by longer PCRs. Adding fresh DNA polymerase helps with enzyme activity lost due to the half life of the polymerase, however this does not help when accurate PCR is required.

It is possible to amplify PCR products up to 20kb using lower or slower heating cycles and special mixtures of polymerases. Long and Accurate PCR is accomplished by using mixtures of processive polymerases and high fidelity Polymerases.