Another method of DNA typing is Short Tandem Repeat (STR), which uses shorter repeat units of DNA nucleotide sequences of 2 to 5 base pair units in length. They are found throughout the chromosomes in vast quantities, increasing the availability for loci to be exposed and verified (NRC, 1996). The loci are run concurrently with an allelic ladder used for direct comparison to alleles found in the population. STR loci are an ideal candidate for degraded DNA because the fragments produced through amplification range from 200-500 base pairs.
Additionally, sample mixtures can be resolved with STR more readily than with previous DNA typing systems (Ruitberg, 2000). The most recent advancement in DNA typing has been the analysis of Mitochondrial DNA (mtDNA). MtDNA can be used to identify bone fragments and strands of hair that contain little or no nuclear DNA that are inherited from maternal lineage. The FBI Laboratory began typing mtDNA in 1996 and has since processed over 500 cases. MtDNA can be advantageous over nuclear DNA given the circumstances that surround many cases and environmental considerations.
First, the location and structure of the mtDNA protects it from environmental degradation and the circular structure of the mitochondria buried within the cell protects it from deterioration. The mtDNA can be found throughout the body in muscles, various cells and bound to hydroxyapatite, an essential ingredient of normal teeth and bones. Second, the high level of mtDNA found throughout the body gives an analyst a better chance at locating a non-degraded sample for amplification (Isenberg, 2002).
It is used by the Armed Forces Institute of Pathology to identify the remains of missing servicemen (Hansen, 1998). More recently many of the victims in the World Trade Center September 11th terrorist attacks were identified using mtDNA analysis due to the gruesomely degraded state of the remains, and in some cases there were only bones available. In cases of missing persons the mtDNA is especially advantageous due its maternal inheritance because a sample from the person’s mother can be stored for future reference in cases of unidentified persons or bodies. DNA Analysis Techniques
After the DNA has been typed it will be analyzed and matched against DNA profiles to determine the sample’s origin. A brief summary of an analysis protocol is to begin with extracting a small segment of DNA from the cells of an individual. If RFLP is used then the DNA in each sample is digested with the same restriction enzymes. Since every person has DNA with slightly different base sequences, some of the restriction sites will be missing or in different locations. Therefore, each person’s DNA restriction enzyme digest will produce unique DNA fragment numbers and sizes.
Then samples of fragmented DNA are placed side by side and ran concurrently with a control DNA and ladder for fragment size, in an agar gel, which separates the samples by size using electrophoresis. The fragments are transferred to a nylon membrane and a radioactively labeled probe comprised of single stranded DNA with complementary bases to the sequence region of interest. The membrane is put on a photographic x-ray film creating an autoradiograph, or autorad, that picks up radiation emitted from the natural decay of the isotope used in your probe.
What is seen on the film is a darkened band that indicates the places on the membrane where the probe has bound to the DNA sequence of interest. In the sexual assault example above a membrane developed with an autorad shows a profile image in which the victim’s DNA and the evidence sperm DNA was ran in comparison to three possible donors of the sperm, one being her boyfriend. In one run this identification ruled out her boyfriend and suspect 2 because they both have a profile different from the semen sample. This is read by how far the fragments ran on the gel, which is determined by size.
Suspect 2’s DNA fragments ran much farther down the gel, meaning that they are shorter, and the boyfriend’s DNA did not run far enough, meaning they are larger. In this sample suspect 1 and the sperm DNA found at the scene match. An added bonus to this profile, incase there were only one suspect, #2 for example, is that it also reveals that the sperm DNA sample came from a heterozygote, because it has two bands of distinct sizes in each lane. In contrast, suspect 2 is a homozygote because there is only one darker band indicating the presence of two copies of the same fragment.
DNA isolated from the victim as well as a human DNA (K562) that served as a control s for standard size references (NRC, 1996). Another technique commonly used with analyzing DNA is the Polymerase Chain Reaction (PCR), which won Dr. Karry Mullis half of the 1993 Nobel Peace Prize in Chemistry. PCR is a method of amplification that has been very effective on samples too minuscule in size or in degradation for reliable results. Generally, PCR is the process of separating and replication repeated, however, the entire procedure only consists of three main steps: denaturation, annealing, and extension.
The first step, denaturation, is the separation of the DNA double helix allowing each strand to be used as a template for the synthesis of new strands. Denaturation is accomplished when the DNA is subjected to high temperatures, 94C. The second step is annealing which involves lowering the temperature to 60i?? C. At this lower temperature the primers are allowed to hybridize the DNA of interest. The final step is extension performed at 70-75 C, and is dependent on the heat-stable Taq polymerase enzyme used for binding DNA for new strands, where replication its highest in this step.
25-40 PCR cycles are usually sufficient to render a reliable test sample. The PCR process is used to replicates defined portions of DNA millions of times over through repeatedly separating paired DNA strands and using each strand as a replication template for a new DNA pair. The PCR product is then analyzed by either sequence polymorphisms or length polymorphisms. The sequence polymorphisms are identified using a hybridization method or direct sequencing and used on samples like mitochondrial DNA.
The length polymorphisms are identified using a gel electrophoresis method (Rudin, 2002). PCR methods are typically coupled with RFLP analysis and have sped the analysis by providing accurate results within 24 hours. Sir Alec Jeffreys also developed a lesser-known system called Minisatellite Variable Repeat Analysis (MVR) for analyzing polymorphisms in 1990, although published, there has not been much work using this method. The MVR system combines the advantages of PCR amplification of sequence-variant alleles with the detection of discrete lengths of VNTR loci.
Briefly, the amplification method occurs in three steps. The first step selects sequence-specific primers and binds them to a small number of repeat complimentary units. In the second step of amplification the molecules are distorted by uniform flanking primers, thus producing a set of representative fragments. The third step amplifies the existing sets of fragments equally using the high concentration of invariant flanking primers with a fourth primer that binds to non-repeat-specific TAG sequences found on the ends of the sequence-specific primer.
The MVR analysis requires the excision of bands out of gels and the use of radioactivity to type the sample. The results are produced as a digital code, ideal in automated systems for detection, analysis and storage. Realistically, this method has not been embraced by the forensic community due to the complicated and labor intensive processing required, and its late introduction that fell after the decisions had been made for choosing a standard amplification system to utilize (Rudin, 2002). In the diagram below a sequence of MVR-PCR maps of 6 class I alleles are presented.
All alleles were MVR mapped with primers that detects both B- and C-type repeats and discriminates between these two variants. The first few repeats were often not detectable after short (14 hr at room temperature) exposure of the autoradiograph. Autoradiographs of each radiolabeled Southern blot were therefore also produced after 36 hr exposure at -80i?? C with intensifier screen. A subdivision of class I alleles is apparent from the autoradiograph presented, most readily defined by the presence or absence of F-type repeats located at the center and top of alleles (Jeffreys, 2000).
Automated Systems are gaining support as many laboratories move toward using “robotics” for mundane tasks like extraction and amplification techniques of DNA, validation of genotypes and band sizing using computer imaging. A typical setup for an automated system to perform DNA extraction and amplification begins with liquid blood transfer from a sample vial with a barcode for identification. An automated pipetter with disposable tips aliquots the blood sample onto a stain card and into a 96-well plate that will be prepared for extraction.
The robot can also prepare stains for long-term storage. A multiple-probe head robot extracts the DNA using an adsorption technique where the blood in each well is passed through a column containing a DNA adsorbing material. This column holds the DNA while non-DNA material is washed out and then the DNA is eluted using a combination of heat and chemicals. This robot can process up to four well plates in around five hours. An automated system can then quantitate the extracted DNA before diluting on a separate plate to be prepped for amplification using a spectrofluorometer.
Another robot performs the amplification using a multi-probe head where it dilutes the samples on a separate plate and then adds a “master mix” comprised of PCR probes and enzymes. The automated system continues with the PCR process (Rudin, 2002). However, the automated process is not completely human free. Quality control steps like ensuring the automated probes are calibrated, scheduled maintenance and validation are all hands-on tasks required ensuring the machines perform accurately.