Technology and Forensic Science

Forensic science is a rapidly growing discipline and the tools available to forensic researchers are also evolving quickly. Painstaking protocols forDNA collection, extraction, quantification, amplification, detection, and analysis have now been replaced by commercially available kits, high-throughput instrumentation, and computer algorithms. With these technologies, forensic scientists have the tools in hand to fortify databases and solve even the most complicated crimes.

There are several steps involved in the process of DNA analysis. These are collection, extraction, quantification, amplification, detection, and analysis. Each step can be accomplished by different methods and can encounter complications. In order to accurately determine a profile from a DNA sample and ensure it is admissible in a court of law, the process must be documented and the protocols followed appropriately.

Sample collection is the first step of processing a forensic sample for DNA analysis. Because contamination can instantly ruin a sample, the investigator at the scene must make cautious efforts not to touch any part of the sample. For example, a blood spot found on an article of clothing at the scene of a crime is easily contaminated with the investigator's DNA if it is picked up by a non-gloved hand. Therefore, forensic investigators and police at crime scenes wear gloves and immediately deposit samples in sealed bags or containers. Scene of crime samples can be in myriad of forms: hair found at the scene, an article of clothing, a piece of chewing gum, or the end of a cigarette, for example. All of these are collected, placed in sterile containers, and brought to the forensic laboratory for investigation.

Samples collected for reference databases are more straightforward than scene-of-crime samples. Reference samples are collected from the offender once he or she is arrested or found guilty. Whether or not a sample can be taken before the alleged perpetrator is convicted in court depends on the country and even the state in which the crime was committed. The most common means of reference sample collection is by buccal swab. This method involves using a sterile swab to wipe the inside of the subject's mouth. DNA is later isolated from the cheek cells attached to the swab. Other methods include drawing blood from the individual and storing it on an FTA® card. These are thick pieces of specialized, sterile paper on which a drop of blood can be absorbed and the DNA can be isolated later. Regardless of the method of collection, all samples are taken to the laboratory for further processing.

When a forensic investigator obtains a sample from which DNA must be analyzed, DNA must first be extracted from the sample. This is the case with either a scene-of-crime sample or a reference database sample. In the case of the latter, DNA extraction is much easier. Commercially available protocols exist for isolating DNA from buccal swabs or FTA® cards. Similarly, many companies now offer kits for the extraction of more complex scene of crime samples. Specialized protocols are available to isolate DNA from trace samples such as cigarette butts or blue jeans. DNA extraction kits are based on one of several different methods. The most basic means is an extraction using phenol-chloroform or alcohol precipitation. These methods are the most common in homemade methods. Kits are also available that use ion exchange resins or silica-based columns to isolate and purify the DNA. One of the newest techniques for DNA isolation involves the use of magnetic beads and specialized buffers. By adjusting the ionic charge of the environment surrounding the sample, DNA will stick to the magnetic beads and can be exposed to a series of buffers to remove contaminants.

A variety of manufacturers are now producing automated machines to aid in the extraction of DNA from forensic samples. Automated systems are available for all different types of chemistries, columns, and magnetic-bead-based extractions described above. There are many benefits to automated DNA extraction; it involves less hands-on time by technicians and thus, has a lower likelihood of contamination or human error with the protocol. Also, automated systems can be very simple and do not require that the laboratory staff be highly trained and specialized. Finally, the use of automated extraction allows for the processing of many more samples a day than manual methods, which can be quite time consuming.

Once the DNA is extracted and quantified, it must be amplified by PCR (polymerase chain reaction) . The PCR method involves three steps: denaturing, annealing, and extension, which are performed in an instrument called a thermalcycler. In more detail, DNA is placed in a reaction tube containing buffers, primers, nucleotides and an enzyme known as Taq polymerase. During the denaturing step, the mixture is subjected to high temperatures so that the double strands of DNA separate. Next, the temperature is lowered to one that allows for annealing of the specific primers to their sequence counterparts on the DNA of the sample. Finally, the temperature in the thermalcycler changes to the optimal temperature for the enzyme Taq polymerase, which extends the regions of DNA between the primers by adding the nucleotides, thus making a copy. The same reaction repeats over many cycles of these three steps resulting in an exponential amplification of the regions between the primers.

The forensic researcher chooses primers for specific sequences of the genome that show regions around the gene sequences that are common amongst individuals. The result of the PCR reaction is then copies of the sequences that differ in the regions between the primers. Several different companies now offer kits that contain all the necessary reagents to amplify STR profiles. Specific kits are even available to amplify only male DNA. The kits also include standards, or specific fragments of DNA of known size to determine the length of each gene sequence. This is important, as the length of the gene sequence is what differs among individuals. The number of repeats in the sequence determines its length. Accumulation of the various repeat regions provides a profile that is specific to a particular person.

In the detection step, the forensic researcher visualizes the DNA. The sequences of the amplified DNA are visualized on a gel that allows for the determination of the number of repeats in the sequence regions. The fluorescent dyes included in some kits help to visualize the sequences. Contemporary forensic scientists tend to use nucleic acid analyzers and automated sequencers for detection of DNA sequences. These automated methods remove the potential subjectivity of the analysis if performed by manual means. The DNA sequences are then entered into a computer and software is used to calculate and store the sequences. Then, sequences of the reference sample or scene-of-crime sample are compared via software programs to other gene sequence profiles in the database. Forensic scientists look for matches to assist them with their investigation.

Automation is commonplace in today's forensic laboratory for the amplification, detection, and analysis steps, as well. Most laboratories performing DNA analysis are equipped with a variety of instruments including extraction systems, thermalcyclers, nucleic acid analyzers, and gene analysis software. What was once was a complex manual process taking days can be accomplished with instruments in only a few hours. Automated methods must be validated, similar to manual methods, to ensure they meet the standards and guidelines set forth by the governing bodies in forensic science, such as Interpol or the Federal Bureau of Investigation.

Following collection, the processes of DNA extraction, quantification, amplification, detection, and analysis can be performed with almost no intervention of the forensic scientist. Automation has revolutionized the forensic laboratory to almost a high-tech factory. With the aid of automated systems, forensic scientists are not only processing their current case samples, but also chipping away at the large backlog of reference samples. DNA databases are building up at a rapid pace, which will ensure that investigators have the best chance of finding a match and solving a crime.

see also DNA; DNA databanks; DNA fingerprint; DNA isolation methods; DNA profiling; Electrophoresis; European Network of Forensic Science Institutes; Interpol; Nucleic Acid Analyzer (HANAA); PCR (polymerase chain reaction); STR (short tandem repeat) analysis; Trace evidence.