Use Of Positive And Negative Controls

As noted by Melton and Nelson (2001) the two primary goals in mtDNA testing are (1) to protect the integrity of the evidence by preventing contamination at any stage in the testing and (2) to collect the maximum amount of available mtDNA data inherent to any sample. Control samples that are processed in parallel with evidentiary samples through each step of the process serve to monitor performance and assess one's success with the two goals noted above.

Contamination assessment is performed with reagent blanks and negative controls. Reagent blanks monitor contamination from extraction to final sequence analysis while negative controls monitor contamination from amplification to final sequence analysis (SWGDAM 2003). All of the procedures performed on a sample are also performed on the reagent or extraction blank with the exception of adding DNA. Negative controls or amplification blanks are introduced at the PCR amplification step and use the same reagents as the sample with sterile water in place of the DNA template. If the reagent blank and/or the negative control associated with a particular amplification results in a sequence that is the same as that of the sample, all data for the sample must be rejected (Isenberg 2004). The analysis must then be repeated beginning with the re-amplification of the sample in question.

Reagent blank contamination is sometimes observed in spite of great efforts to keep the laboratory environment clean. Since mtDNA analysis is a very sensitive technique, the presence of low-level contamination is not uncommon (Isenberg 2004). For example, Mitotyping Technologies reported that reagent blanks resulted in amplification products in 29 of 1218 (2.4%) of PCR reactions performed in casework over a two year period of time (Melton and Nelson 2001). These contaminants did not match a staff member's type or the type of the recently handled sample a fact that suggests they are likely sporadic contaminants to a particular disposable tip or PCR tube. This type of contamination is not uncommon when working with low-copy number DNA as noted in Chapter 7.

If contamination is observed with either the reagent blank or the negative control, results from the unknown sample being run in parallel do not always have to be disregarded. Research with artificial sample mixtures has demonstrated that a threshold of background contamination can be set for still obtaining reliable sequence data. For example, the FBI Laboratory has established a 10:1 rule where any contamination seen in a reagent blank or negative control during post-PCR analysis must be less than one-tenth the amount of the sample being processed (Wilson et al. 1995a, 1995b). This sample-to-contamination ratio determination is possible due to the PCR product quantification analysis performed in their procedure. A more recent study demonstrated that the 10:1 rule is conservative and reliable (Stewart et al. 2003).

A positive control is a sample of known mtDNA sequence that serves to demonstrate that amplification and sequencing reaction components are working properly. This positive control is typically an extracted DNA sample that is processed through the steps of amplification, sequencing, and data analysis. For example, the FBI Laboratory uses the HL60 cell line as a positive control.

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