Pcr Inhibitors And Dna Degradation

When extracting biological materials for the purpose of forensic DNA typing, it is important to try and avoid further degradation of the DNA template as well as to remove inhibitors of the polymerase chain reaction (PCR) where possible. The presence of inhibitors or degraded DNA can manifest themselves by complete PCR amplification failure or a reduced sensitivity of detection usually for the larger PCR products (see Chapter 7).

Two PCR inhibitors commonly found in forensic cases are hemoglobin and indigo dyes from denim. Melanin found in hair samples can be a source of PCR

Type of Sample

Amount of DNA

Liquid blood

20 000-40 000 ng/mL

Blood stain

250-500 ng/cm2

Liquid semen

150 000-300 000 ng/mL

Post-coital vaginal swab

10-3000 ng/swab

Plucked hair (with root)

1-750 ng/root

Shed hair (with root)

1-10 ng/root

Liquid saliva

1000-10 000 ng/mL

Oral swab

100-1500 ng/swab


1-20 ng/mL


3-10 ng/mg


Typical DNA amounts that may be extracted from biological materials (Lee and Ladd 2001). Both quality and quantity of DNA recovered from evidentiary samples can be significantly affected by environmental factors.

inhibition when trying to amplify mitochondrial DNA (see Chapter 10). These inhibitors likely bind in the active site of the Taq DNA polymerase and prevent its proper functioning during PCR amplification.

DNA degrades through a variety of mechanisms including both enzymatic and chemical processes (Lindahl 1993). Once a cell (or organism) dies its DNA molecules face cellular nucleases followed by bacterial, fungal, and insect onslaughts depending on the environmental conditions (Poinar 2003). In addition, hydrolytic cleavage and oxidative base damage can limit successful retrieval and amplification of DNA. The main target for hydrolytic cleavage is the glycosidic base sugar bond. Breakage here leads to nucleobase loss and then a single stranded 'nick' at the abasic site. If a sufficient number of DNA molecules in the biological sample break in a region where primers anneal or between the forward and reverse primers, then PCR amplification efficiency will be reduced or the target region may fail to be amplified at all. Thus, heat and humidity, which speed up hydrolytic cleavage, are enemies of intact DNA molecules. Furthermore, UV irradiation (e.g., direct sunlight) can lead to cross-linking of adjacent thymine nucleotides on the DNA molecule, which will prevent passage of the polymerase during PCR.


To ensure that DNA recovered from an extraction is human rather than from another source such as bacteria, DNA Advisory Board standard 9.3 requires human-specific DNA quantitation (see Appendix IV). Only after DNA in a sample has been isolated can its quantity and quality be reliably assessed. Determination of the amount of DNA in a sample is essential for most PCR-based assays because a narrow concentration range works best. For example, the Applied Biosystems' Profiler Plus™ and COfiler™ multiplex STR kits specify the addition of between 1-2.5 ng of template DNA for optimal results (Applied Biosystems 1998). Promega STR kits also work best in a similar DNA concentration range (Krenke et al. 2002). Too much DNA can result in split peaks or peaks that are off-scale for the measurement technique (see Chapter 6). Too little DNA template may result in allele 'drop-out' because the PCR reaction fails to amplify the DNA properly. This phenomenon is sometimes referred to as stochastic fluctuation (see Chapter 4).

Early methods for DNA quantitation typically involved either absorbance at a wavelength of 260 nm or fluorescence after staining a yield gel with ethidium bromide. Unfortunately, because these approaches are not very sensitive, they consume valuable forensic specimens that are irreplaceable. In addition, absorbance measurements are not specific for DNA and contaminating proteins or phenol left over from the extraction procedure can give falsely high signals.

To overcome these problems, several methods have been developed for DNA quantitation purposes. These include the slot blot procedure and fluorescence-based microtiter plate assays as well as so-called 'real-time or quantitative PCR' approaches. A nice review of various DNA quantification methods was recently published (Nicklas and Buel 2003b).

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