A forensic DNA laboratory often has to deal with DNA samples that are less than ideal. The biological material serving as evidence of a crime may have been left exposed to a harsh environment for days, months, or even years, such as in the case of an investigation into a missing person. The victims of homicides are typically taken to out of the way places where they remain until their bodies are discovered. Instead of being preserved in a freezer away from caustic chemicals that can break it down, the DNA molecules may have been left in direct sunlight or in damp woods.
Regardless of the situation, the DNA molecules from a crime scene come from a less than pristine environment that is normally found in molecular biology laboratories. Just as important is the fact that the retrieved biological sample may be limited in quantity. Thus, accurate sample analysis is critical since a forensic scientist may only obtain enough evidence for one attempt at analysis. In this chapter, we explore the forensic issues surrounding the analysis of short tandem repeats (STRs) including handling degraded DNA samples, avoiding contamination, overcoming polymerase chain reaction (PCR) inhibition, and interpreting mixtures, which are prevalent in forensic cases especially those involving sexual assault. In addition, we discuss the use of low-copy number DNA profiling to retrieve genetic information from samples with only a few cells available for testing.
Environmental exposure degrades DNA molecules by randomly breaking them into smaller pieces. Enemies to the survival of intact DNA molecules include water and enzymes called nucleases that chew up DNA. Both are ubiquitous in nature. With older technologies such as restriction fragment length polymorphism (RFLP), these severely degraded DNA samples would have been very difficult if not impossible to analyze. High molecular weight DNA molecules need to be present in the sample in order to detect large VNTR (variable number of tandem repeats) alleles (e.g., 20 000 bp) with RFLP techniques.
An ethidium-bromide stained agarose 'yield gel' may be run to evaluate the quality of a DNA sample. Typically high molecular weight, high quality genomic DNA runs as a relatively tight band of approximately 20 000 bp relative to an appropriate molecular weight marker. On the other hand, degraded DNA appears as a smear of DNA that is much less than 20 000 bp in size (Figure 7.1a).
Modern-day PCR methods, such as multiplex STR typing, are powerful because miniscule amounts of DNA can be measured by amplifying them to a level where they may be detected. Less than 1 ng of DNA can now be analyzed with multiplex PCR amplification of STR alleles compared to 100 ng or more that might have been required with RFLP only a few years ago. However, this sensitivity to low levels of DNA also brings the challenge of avoiding contamination from the police officer or crime scene technician who collects the biological evidence.
In order for PCR amplification to occur, the DNA template must be intact where the two primers bind as well as between the primers so that full extension can occur. Without an intact DNA strand that surrounds the STR repeat region to serve as a template strand, PCR will be unsuccessful because primer extension will halt at the break in the template. The more degraded a DNA sample becomes, the more breaks occur in the template and fewer and fewer DNA molecules contain the full length needed for PCR amplification.
Was this article helpful?
This book discusses the futility of curing stammering by common means. It traces various attempts at curing stammering in the past and how wasteful these attempt were, until he discovered a simple program to cure it. The book presents the life of Benjamin Nathaniel Bogue and his struggles with the handicap. Bogue devotes a great deal of text to explain the handicap of stammering, its effects on the body and psychology of the sufferer, and its cure.