usually affect PCR through interaction with DNA or interference with the DNA polymerase
. 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 Mg<sup>2+</sup>) or otherwise interfering with their interaction with the DNA polymerase, PCR is inhibited.
In a multiplex PCR
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
, 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... Read More