Archived | DNA Extraction and Quantitation for Forensic Analysts | Inhibitors

Image of inhibitors — National Institute of Justice (NIJ) (see reuse policy).

It is hard to determine all of the causes of inhibition on the PCR reaction. The PCR process can be affected by compounds that interfere with the interaction between DNA and Taq polymerase, and thus inhibit the reaction. Many inhibitors are removed during the extraction process through ethanol precipitation or the use of a Microcon^® or Centricon^® centrifugal filter unit. However, some inhibitors co-elute with the DNA, which may lead to PCR inhibition. A number of inhibitors are contained in the samples themselves, while others can be introduced by the substrate or the analysis process. The presence of inhibitors can result in loss of data or results that could be mistaken for degradation. We can classify sources of inhibition into three groups:

Internal, or those found in body fluids.
Substrates, or those arising from the materials on which the blood stain or other source of DNA has been deposited.
Other sources, such as reagents and materials used in the analysis.

Internal

Many body fluids contain substances that can inhibit PCR:⁰¹

Table of Biological Substances and Inhibitors
*Biological Substance*	*Inhibitor*	*Comment*
Blood	Heme Immunoglobulin G	The amplification of blood samples can be significantly reduced or blocked by natural components of blood, such as heme, and immunoglobulin G. Hemin, a hemoglobin derivative, and its breakdown products, bilirubin and bile salts, are also found to be PCR inhibitors.
Vaginal Samples	Bacteria Microorganisms	Bacteria and microorganisms are commonly found in vaginal, fecal, and buccal samples. Note: these can also be found in other biological samples found at crime scenes.
Buccal Samples
Fecal Samples
Hair Tissue	melanin	Melanin, a pigment that affects skin, eye, and hair color, can inhibit PCR.
Bone Teeth	Ca²⁺	Ca²⁺ is commonly found in bone and teeth and is known to interfere with Mg²⁺ concentration, which in turn may affect the activity of Taq polymerase.
Semen	Polyamines	Spermine and spermidine (polyamines originally isolated from semen) are found in ribosomes and living tissues, and can inhibit PCR.
Urine	Urea	Urea, a chemical found in urine, can inhibit PCR.

Substrates

Some of the substrates that contain inhibitors are:

Table of Substrates and Inhibitors
Substrate	Inhibitor	Comment/Example
Textile dyes	Textile dyes	Indigo dye used to color denim
Fabrics	Tannic Acid	Leather
Environmental Samples	Humic compounds Heavy metals	Soil⁰²
Food Constituents	Organic compounds Phenolic compounds Glycogen Fats Ca²⁺	Many food products, such as milk, contain inhibitors, like Ca²⁺. However, forensic scientists have had success in developing DNA profiles from saliva left on food or drink containers.⁰¹

Examples of substrates with inhibitive properties

National Institute of Justice (NIJ) (see reuse policy).

Other Inhibitors

Inhibitors can be introduced in the collection and analysis processes or at the crime scene

Table of Other Inhibitors
Source	Inhibitor	Comment
Extraction Chemicals	Phenolic compounds from the organic extraction Chelex resin Salts Guanidine Proteases Organic solvents Phosphate buffers Detergents (such as Sodium Dodecyl Sulfate (SDS))	Reagents commonly used in the purification of nucleic acids are inactivators of DNA polymerases. Phenol or Chelex resin left with the extracted DNA can inhibit the PCR process.
Anticoagulants	EDTA and heparin	Known blood reference samples are collected in tubes containing anticoagulants.
Powder	Glove powder	Many forensics scientists use powdered gloves.
Laboratory plastic ware	PCR tubes	It has been reported that an inhibitory substance can be released from polystyrene or polypropylene upon exposure to ultraviolet light.
Plant and food products	Pollen Cellulose Plant polysaccharides Ca²⁺	Biological material can be deposited on plants and food.

Back Forward

Additional Online Courses

See additional NIJ-sponsored training

Date Created: July 6, 2023