PCR inhibitors are any factor which prevent the amplification of nucleic acids through the

polymerase chain reaction The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) ...

(PCR). PCR inhibition is the most common cause of amplification failure when sufficient copies of DNA are present. PCR inhibitors usually affect PCR through interaction with DNA or interference with the

DNA polymerase A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create ...

. Inhibitors can escape removal during the DNA purification procedure by binding directly to single or double-stranded DNA. Alternatively, by reducing the availability of cofactors (such as Mg²⁺) or otherwise interfering with their interaction with the DNA polymerase, PCR is inhibited. In a

multiplex PCR Multiplex polymerase chain reaction (Multiplex PCR) refers to the use of polymerase chain reaction to amplify several different DNA sequences simultaneously (as if performing many separate PCR reactions all together in one reaction). This process ...

reaction, it is possible for the different sequences to suffer from different inhibition effects to different extents, leading to disparity in their relative amplifications.

Types of inhibitors

Inhibitors may be present in the original sample, such as blood, fabrics, tissues and soil but may also be added as a result of the sample processing and DNA extraction techniques used. Excess salts including KCl and NaCl, ionic detergents such as sodium deocycholate, sarkosyl and SDS, ethanol, isopropanol and phenol among others, all contribute via various inhibitory mechanisms, to the reduction of PCR efficiency.

Quantifying extent of inhibition

In order to try to assess the extent of inhibition that occurs in a reaction, a control can be performed by adding a known amount of a template to the investigated reaction mixture (based on the sample under analysis). By comparing the amplification of this template in the mixture to the amplification observed in a separate experiment in which the same template is used in the absence of inhibitors, the extent of inhibition in the investigated reaction mixture can be inferred. Of course, if any part of the inhibition occurring in the sample-derived reaction mixture is sequence-specific, then this method will yield an underestimate of the inhibition as it applies to the investigate sequence(s).

Preventing PCR inhibition

Sample collection

The method of sample acquisition can be refined to avoid unnecessary collection of inhibitors. For example, in forensics, swab-transfer of blood on fabric or saliva on food, may prevent or reduce contamination with inhibitors present in the fabric or food.

DNA purification

Techniques exist and kits are commercially available to enable extraction of DNA to the exclusion of some inhibitors.

PCR reaction components

As well as methods for the removal of inhibitors from samples before PCR, some DNA polymerases offer varying resistance to different inhibitors and increasing the concentration of the chosen DNA polymerase also confers some resistance to polymerase-targeted inhibitors. For PCR based on blood samples, the addition of

bovine serum albumin Bovine serum albumin (BSA or "Fraction V") is a serum albumin protein derived from cows. It is often used as a protein concentration standard in lab experiments. The nickname "Fraction V" refers to albumin being the fifth fraction of the origin ...

reduces the effect of some inhibitors on PCR.

References

{{DEFAULTSORT:Polymerase Chain Reaction Inhibitors DNA Polymerase chain reaction