DNA Typing Systems
Deoxyribonucleic acid (DNA) typing is a way to categorize an individual's genetic makeup in order to distinguish one individual from another. This has been made possible due to the rapid acceleration of genomics-based technologies coupled with the fact that human genomic DNA, which is comprised of 3 billion bases (letters in the DNA alphabet), is unique in only 0.1% of its makeup. Therefore, approximately 3 million bases differ from one person to the next, allowing scientists to use these differences to perform identity matches with a high degree of certainty. These variable regions of DNA can be used to generate a DNA profile of an individual, using samples from blood, bone, hair, semen, and saliva, as well as other body tissues.
In DNA typing, there are several systems that can be employed to characterize DNA from a sample. These systems have different applications and purposes. For example, in forensics, scientists may need to obtain DNA from a crime scene in order to analyze a specific set of DNA markers (regions within the genome that are variable) rapidly, yet with good results. DNA typing systems that have previously been used or are currently being used in forensics include restriction fragment length polymorphism (RFLP) typing, short tandem repeat (STR) typing, single nucleotide polymorphism (SNP) typing, mitochondrial DNA (mtDNA) analysis, human leukocyte Antigen (HLA) typing, gender typing, and Y-chromosome typing. RLFP analysis was the first major DNA typing system used in forensics. All of the techniques that followed could not have been developed without the discovery of a revolutionizing methodology called the polymerase chain reaction, or PCR. PCR allows a scientist to amplify genomic DNA (small sequences up to a few thousand in length) extracted from a sample so that there are sufficient quantities to be analyzed.
RFLP analysis was first developed by Alec Jeffreys. RFLPs can be used to analyze the DNA directly in a way that is fairly inexpensive. In RFLP analysis, genomic DNA is digested with a molecular enzyme that cuts the DNA at specific sequences it recognizes, creating multiple fragments. These fragments can be variable depending on whether the enzyme cuts at a particular site in the DNA. Variable DNA that is inherited (and not mutated) at a site may or may not be cut by the enzyme depending on whether the sequence contains the enzyme recognition site. These sites can be highly variable based on inheritance patterns. For this reason, a pattern of fragments will be produced based on the number of cut sites, separated by gel electrophoresis, transferred to a nitrocellulose membrane, and using radio-labeled probes (short sequences of DNA that bind to the complementary sequence from the sample DNA) that bind to specific sequences of interest, they can be identified by audioradiography. Radioactively labeled probes are visualized by audioradiography, or what appears to be film that has burned bands, based on size, that run in the gel during gel electrophoresis. If there is a lack of a restriction site in an individual's DNA at a specific site, the enzyme will not cut it and the fragment will therefore be larger. RFLP analysis is not always applicable because it requires a large amount of high quality DNA. In forensics, samples obtained from a crime scene tend to be degraded. Although RFLP is one of the original applications of DNA analysis that forensic investigators used, newer, more efficient DNA-analysis techniques have replaced this technology.
PCR-based assays followed RFLP analysis because of their greater sensitivity, simplicity, and amenability to analyzing degraded DNA samples. PCR can amplify extremely small amounts of DNA (even DNA from a single cell) to large DNA concentrations (nanograms). Using PCR to amplify a specific sequence of interest, which contains a variable sequence within the amplicon (amplified PCR fragment), STR analysis can be performed. STRs are short tandem repeats of 2–5 base pairs that are repeated a few to dozens of times. Identification of the STR can be performed by direct DNA sequencing. However, it is most often analyzed using gel electrophoresis (if the difference in tandem repeat is large enough) with ethidium bromide, a carcinogen that inserts DNA and fluoresces with an ultraviolet lamp. As the size of the amplified STR loci is in the range of 200–500 base pairs, it makes it ideal for degraded DNA samples.
The Federal Bureau of Investigation (FBI) uses a set of thirteen specific STR regions for CODIS, a software program that comes from a database derived from local, state, and national agencies using information collected from criminals or arrested individuals. With these markers, it is estimated that there is approximately a one in one billion chance that two individuals will be the same at the thirteen different marker sites.
Another DNA typing system, which is used most frequently in forensics, is mitochondrial DNA analysis. Mitochondrial DNA (mtDNA) is DNA that comes from a source separate from the DNA found in the nucleus. It is much smaller (only 16.5 thousand bases) than nuclear DNA and is important for producing proteins that are important and specific to energy production within the cell. The advantage of using mtDNA is because many tissues (such as muscle) have a much higher copy number of mtDNA compared to nuclear DNA, which only has two copies of genetic information and two sex chromosomes. For samples with little DNA recovered, mtDNA analysis is the preferred approach. It is also important for samples that do not have nucleus, such as red blood cells, rootless hair, bones, nail clippings, and teeth. For these tissues, STR and RFLP analysis cannot be used. Finally, mtDNA analysis is possible due to a highly variable region (by 1–2% in unrelated individuals) in the mtDNA genome called the "D-loop." Mitochondrial DNA is maternally inherited.
Y-chromosome analysis is only applicable in cases that test for identity matches in males. Y-chromosomes can only be inherited by sons from fathers. It can also be useful in testing male suspects when multiple sample sources have been identified at a crime scene. Gender typing can also be performed by analyzing the X-chromosome and determining if there is one allele (male) or two different alleles (female) at an informative site.
Another DNA typing system, used in particular by forensic scientists, involves designing small pieces of short DNA sequences called "probes" that bind to complementary DNA sequences extracted from a sample found at a crime scene. Much like the radiolabeled probes, these short sequences can be used to create a distinct pattern depending on the DNA source. These patterns can be compared to the sample from a crime scene and determined if a match exists between the DNA from the sample and the DNA from the suspect. These probes can be fluorescently labeled and used to identify Small Nucleotide Polymorphisms (SNPs), which are single base variations that are known to be variable within a given population, are not themselves disease-causing, do not represent spontaneous mutations, and are found throughout the genome. A marker is only informative if there is a difference between two samples. Although a single SNP may not be informative, combining several SNPs is useful and can easily be automated. The more markers that are used, the likelihood that the two samples are identical is greater.
Although six or more probes are usually used in forensics DNA typing, new, more advanced DNA typing systems are being developed. DNA-chip technology is the latest molecular advancement that will considerably speed up analysis and allow forensic scientists to study many sequences at one time in a fully automated manner. DNA chips (also known as microarrays) have small sequences printed or synthesized onto microscopic spots on a tiny chip. When DNA is added to the chip, binding of the sample to the probe occurs when there is a match. The probes are labeled with a fluorescent dye that fluoresces when it hybridizes to a sequence from the sample DNA. Different fluorochromes (colors) can be used to distinguish which DNA base is present in a variable position of the DNA sequence.
Despite its speed, this type of DNA technology is more expensive and probably better suited for applications where a large number of suspects' samples are required for DNA typing. It might also apply DNA typing to identify the remains of many different individuals from a natural disaster. For example, after the tsunami that developed off the west coast of the Indonesian islands in 2004, coastal regions in Thailand and other Asian countries were devastated by its destruction. Many visitors' and residents' remains were found but could not be identified. Microarray-based DNA typing would have been helpful in characterizing the DNA patterns from the remains and matching them to various samples.
After the discovery of PCR, a DNA typing system that was used in forensics was the HLA DQ a / HLA DQA1 system, or Human Leukocyte Antigen (HLA) system. This system is comprised of a 242 base area in the genome that is highly variable in the population. It can be detected using molecular probes that seek out complementary subregions within this genetic region. The probe for HLA DQa set started out with six common sites called DQ alleles that, when combined, produced 21 possible genotypes. With only one locus or region in the genome used, the predictive value was lower than RFLP analysis or other PCR-based assays. The HLA DQA1 system improved the analysis by detecting 28 possible genotypes. The AmpliType PM+DQA1, another HLA-related locus, was developed to expand the HLA DQ system. It uses several markers at different loci (the location of a particular gene on a chromosome), analyzed simultaneously (also called multiplexing). With five additional markers analyzed, the statistical power increases considerably.