Solutions To Pcr Inhibition

A nice review of strategies to generate PCR-compatible samples was published recently (Radstrom et al. 2004, see also Wilson 1997). PCR inhibitors may be removed or their effects reduced by one or more of the following solutions. The genomic DNA template may be diluted, which also dilutes the PCR inhibitor, and re-amplified in the presence of less inhibitor. Alternatively, more DNA polymerase can be added to overcome the inhibitor. With this approach some fraction of the Taq polymerase binds to the inhibiting molecule(s) and removes them from the reaction so that the rest of the Taq can do its job and amplify the DNA template. In addition, polymerases other than Taq have been shown to work well with blood and feces, which typically inhibit PCR when performed with Taq DNA polymerase (Al-Soud and Radstrom 1998).

Additives to the PCR reaction, such as bovine serum albumin (BSA) (Comey et al. 1994) or betaine (Al-Soud and Radstrom 2000), have been shown to prevent or minimize the inhibition of PCR. More recently a sodium hydroxide treatment of DNA has been shown to neutralize inhibitors of Taq polymerase (Bourke et al. 1999). The addition of aluminum ammonium sulfate proved helpful to prevent the co-purification of inhibitors with DNA from soil samples (Braid et al. 2003). Finally, a separation step may be performed prior to PCR to separate the extracted DNA from the inhibiting compound. Centricon-100 and Microcon-100 filters have been used for this purpose (Comey et al. 1994) as have low-melt agarose gel plugs (Moreira 1998).

CONTAMINATION ISSUES

The sensitivity of PCR with its ability to amplify low quantities of DNA can be a problem if proper care is not taken. Validated laboratory protocols must be adhered to so that contamination from higher concentrations of DNA, such as those of the DNA analyst, can be avoided. However, it is important to keep in mind that if contamination does occur, it will most likely result in an 'exclusion' or 'inconclusive' result and be in favor of the defendant (see Chapter 15).

Contamination implies the accidental transfer of DNA. There are three potential sources of contamination when performing PCR: sample contamination with genomic DNA from the environment, contamination between samples during preparation, and contamination of a sample with amplified DNA from a previous PCR reaction (Lygo et al. 1994). The first source of contamination is largely dependent on sample collection at the crime scene and the care taken there by the evidence collection team (see Chapter 3). Environment contamination can be monitored only in a limited sense by 'substrate controls' (Gill 1997). The latter two sources of contamination can be controlled and even eliminated by using appropriate laboratory procedures and designated work areas (see Chapter 4).

The possibility of laboratory contamination is addressed with 'negative controls' that test for contamination of PCR reagents and tubes. Basically, a negative control involves running a blank sample through the entire process in parallel with the forensic case evidence. The same volume of purified water as DNA template in the other samples is added to a negative control PCR reaction. If any detectable PCR products are observed in the negative control, then sources of contamination should be sought out and eliminated before proceeding further.

In a systematic examination of possible sources of PCR contamination, Henry Lee and co-workers found that under the circumstances normally encountered during casework analysis, PCR contamination was never noted (Scherczinger et al. 1999). Using reverse dot blot detection of AmpliType PM and DQA1 systems (the same PCR principles apply as with STR markers detected using fluorescence), they examined four general aspects of PCR during which contamination might occur: PCR amplification setup, handling the PCR products, aerosolization, and DNA storage. In these studies, detectable contamination occurred only when gross deviations from basic preventative protocols were employed. They concluded that contamination could not be generated by simple acts of carelessness (Scherczinger et al. 1999).

The genotypes of laboratory personnel are typically stored for comparison purposes so that any contamination by an individual in the lab can be picked up. This 'staff elimination database' is often searched prior to concluding that a generated DNA profile accurately reflects the evidence and is not due to contamination from laboratory personnel (Howitt et al. 2003). Only in the case of gross laboratory error, would a mixture of two DNA types result (i.e., that of the analyst and the sample being examined). These types of errors would most likely be sorted out by comparing the result with genotypes of laboratory personnel when the mixture analysis is performed (see discussion on mixtures below). Some expert system software has been developed to aid detection of potential cross-contamination within a batch of samples (see Chapter 17).

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Stammering Its Cause and Its Cure

Stammering Its Cause and Its Cure

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