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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 commonly included in STR typing kits as a more general means of overcoming the effect of enzyme inhibitors.
Quantitative PCR gives analysts the ability to assess inhibition during the quantitation process. However, not all inhibition will be detected.
Read more in the quantitation module in this course.
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 reamplify. 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
Reextraction & Additional Sample Preparation
An analyst can take additional steps to clean up the sample by reextraction using Chelex resin, phenol chloroform, Thiopropyl Sepharose 6B (Sigma)04 extraction beads, silica based method 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 effects 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.
Additional Online Courses
- What Every First Responding Officer Should Know About DNA Evidence
- Collecting DNA Evidence at Property Crime Scenes
- DNA – A Prosecutor’s Practice Notebook
- Crime Scene and DNA Basics
- Laboratory Safety Programs
- DNA Amplification
- Population Genetics and Statistics
- Non-STR DNA Markers: SNPs, Y-STRs, LCN and mtDNA
- Firearms Examiner Training
- Forensic DNA Education for Law Enforcement Decisionmakers
- What Every Investigator and Evidence Technician Should Know About DNA Evidence
- Principles of Forensic DNA for Officers of the Court
- Law 101: Legal Guide for the Forensic Expert
- Laboratory Orientation and Testing of Body Fluids and Tissues
- DNA Extraction and Quantitation
- STR Data Analysis and Interpretation
- Communication Skills, Report Writing, and Courtroom Testimony
- Español for Law Enforcement
- Amplified DNA Product Separation for Forensic Analysts