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DNA Extraction and Quantitation for Forensic Analysts

Inhibitors

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Image of inhibitors
National Institute of Justice (NIJ) (ver política de reutilización).

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
Examples of substrates with inhibitive properties
National Institute of Justice (NIJ) (ver política de reutilización).

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.

 

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