Overcoming Inhibition

Overcoming Inhibition

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Not all of the factors affecting inhibition are known, and most of the methods used to overcome inhibition are specific to the inhibiting compound. In contrast, Bovine serum albumin (BSA) is included in both Applied Biosystems and Promega STR typing kits as a more general means of overcoming the effect of enzyme inhibitors.

Amplification test gels are a long-established technique to evaluate samples for inhibition. More recently, Quantitative PCR gives analysts the ability to assess inhibition during the quantitation process. However, not all inhibition will be detected. One certain way to assess a sample for inhibition is to add a portion of the extract to a control sample and attempt amplification. Failure of amplification demonstrates the presence of an inhibitor.

Click here to read more about quantitation in Subject 03, Module 05.

The following are techniques that can be used to overcome inhibition:

Dilution

One of the easiest approaches taken to overcome inhibition is to dilute the template DNA sample and re-amplify. This dilutes out the inhibitor, allowing successful amplification to occur. In general, the quality of DNA and purity of the sample is more important to amplification than the quantity of DNA. Usually, a 1:10 or greater dilution is sufficient to overcome inhibition.

Bovine Serum Albumin (BSA)

BSA has been shown to reduce inhibition in samples affected by hemin as well as other inhibiting compounds. BSA is a general stabilizing agent, as is gelatin, which has also been used to overcome inhibition. 03, 02, 04

Re-extraction & Additional Sample Preparation

An analyst can take additional steps to clean up the sample by re-extraction using Chelex resin, phenol chloroform, Thiopropyl Sepharose 6B (Sigma) 04 extraction beads, or magnetic beads (DNA IQ™). Sample preparation using a Centricon^® or Microcon^® device may also assist in removing inhibitors.

Increased Taq Polymerase

The interaction between Taq polymerase and the template DNA is affected by inhibitors. Increasing the concentration of Taq polymerase in the reaction can overcome the affects of some inhibitors. 04

Non-Ionic Detergents

Non-ionic detergents, such as Tween^®-20, NP-40 and Triton^® X-100, can assist in overcoming inhibition, having the specific benefit of overcoming the inhibitory effects of trace amounts of strong ionic detergents, such as SDS. 05

Miscellaneous Chemicals

Ammonium ions and dimethyl sulfoxide (DMSO) have been used to overcome inhibition.

Sodium Hydroxide (NaOH)

One study cites a method for neutralization of Taq polymerase inhibitors by denaturing and washing with NaOH in Microcon^®-100 filtration units. The study speculates that many inhibitors bind the template DNA and possibly intercalate into the double stranded DNA. Denaturizing the DNA could reduce the substance’s affinity for it. One other hypothesis is that the NaOH may directly inactivate the inhibitor.06

Mini Primer Sets

The effects of some inhibitors can be overcome with the use of shorter primers.07

Summary

This module has briefly dealt with collection of DNA from scenes, and with one of the main difficulties encountered with the PCR process, namely the presence of inhibitors. The module describes the nature of inhibitors and techniques for their removal or neutralization.

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