Basic Overview of Forensic DNA and Genophiler®

Forensic DNA and STRs

Approximately 99% of all human DNA is the same. In the one percent that differs, sequences of repeating nucleotides exist called Short Tandem Repeats (STRs). STR regions are highly polymorphic, meaning that they are very different between any number of people. The FBI has marked 13 locations (or loci) on the DNA for forensic testing and identification. 

DNA analysis is an elaborate process. Once a DNA sample has been obtained, it is amplified using Polymerase Chain Reaction (PCR). PCR works as a sort of Xerox machine, making copies of the DNA sample in a lab. STR-containing regions are specifically amplified in a way that labels each with one of four different dyes, then separated and placed in a genetic analyzer, such as the ABI 310 or 3100. The machine uses capillary electrophoresis to separate the STR segments by size, and measures their presence with a laser by recording the fluorescence of light. The results are stored as raw electronic data.
Figure 1: Genotyper® output illustrating the presence of alleles, shown as peaks. The graph is broken into three sections, representing three different DNA locations (loci).

A CD of raw DNA sample data is not very useful because it requires further analysis. The current standard in analysis software is the Applied Biosystems (ABI) GeneScan® and GenoTyper® software packages. GeneScan®identifies the signal corresponding to each dye present in the sample. Genotyper® uses the GeneScan® analysis to identify and label each peak. Operating the GeneScan® and Genotyper® software is not necessarily difficult, but does require special training and is time consuming. DNA profiling experts often spend between five and ten hours to analyze the raw data in a case before even determining if important issues need to be addressed in greater detail.

The GeneScan® and Genotyper® programs produce a considerable amount of output. Genotyper® produces the final output in the form of an electropherogram, a graph that looks similar to an EKG (see Figure 1). The graph is mostly flat with peaks representing the presence of DNA markers (alleles). DNA experts evaluate the output to identify problems and match multiple samples.
Genophiler® is an automation tool used to operate GeneScan® and Genotyper® and collect the resulting data. Genophiler® is essentially a macro that has programmed all of the same mouse clicks and keystrokes that a trained person would use to operate the analysis software. Genophiler® creates PDFs of all output screens to allow for a summarized review of the electronic data without the underlying analysis software. In addition, Genophiler®creates summary tables containing the labeled peaks for each tested sample and an automated report containing a list of potential issues that may require further attention (such as the presence of possible mixtures, peak height imbalances, pull-up, etc.). These potential issues are identified using generally-accepted methods and all methods are explained in the Genophiler® outputs.
Genophiler® has been developed from the ground up to be completely transparent. Every action is fully documented. In addition, all data files are provided, including sample files, size standards, analysis parameters, GeneScan®projects, and Genotyper® projects with macros. Anyone who knows how to operate GeneScan® and Genotyper® can replicate any or all of the Genophiler® analysis.
Genophiler® has been used to automate the electronic analysis of forensic DNA sample data in more than 700 cases. Genophiler® has been extensively validated since its development and it continues to be evaluated with every case that we review.

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