DNA is the material within every cell of the body and represents the blueprint of life. It allows physical traits to be passed on from one generation to the next. Although the majority of the human genome (the complete set of genes for an individual) is the same across all ethnic populations, people differ in their genetic makeup by a minuscule amount, and thus have their own unique DNA pattern. DNA profiling, also referred to as DNA typing, is the molecular genetic analysis that identifies DNA patterns. In forensic science, DNA profiling is used to identify those who have committed a crime. It is estimated that roughly one percent of all criminal cases employ this technique; however, DNA profiling has been used to acquit several suspects involved in serious crimes such as rape and murder and it has been used to convict individuals of crimes years after investigators closed the unsolved case. Aside from identifying an individual responsible for violent crimes, the judicial system also can use DNA profiling to determine family relationships in the case of disputed paternity or for immigration cases.
DNA molecular analysis has also been used in the diagnosis of clinical disorders. Many genetic diseases are caused by mutations in DNA within regions of the genome that code for protein, and scientist look in these regions for mutations to determine if a patient is affected or is a carrier of a genetic disease. Unlike clinical molecular genetics, DNA typing for forensics takes advantage of locations within the human genome that do not code for protein. These locations typically involve repetitive DNA sequences that are polymorphic, or have a variable number of repeat sizes. Because non-protein-coding DNA is used, DNA databanks that contain DNA typing information do not reveal any information about an individual's health status or whether the individual has or is a carrier of a genetic disease.
The sensitivity of DNA profiling tests have dramatically increased over the last two decades. It used to be necessary to have a sample roughly the size of the ink in an ink pen, skilled forensic scientists can now obtain enough DNA from saliva left on the end of a cigarette to get a DNA profile result. The speed at which results can be obtained has also dramatically improved. This is all, in part, due to the discovery of the polymerase chain reaction, a technique that can amplify large amounts of specific small sequences of DNA from the human genome. It is also due to the advent of various DNA fingerprinting tools. The effect of these advances has broadened the sample size and quality required for analysis.
DNA profiling uses a variety of DNA typing systems, including: 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.
The first approach to DNA typing used variable number tandem repeats, or VNTRs. VNTR's are repeating units of a DNA sequence, the number of which varies between individuals. They are analyzed as Restriction Fragment Length Polymorphisms (RFLPs). RFLPs are variations within specific regions of genomes that are detected by restriction enzymes. RFLP analysis originated in the 1970s after the discovery of restriction enzymes, or proteins that can cut DNA into smaller molecules (restriction fragments) based on specific DNA sequence recognition sites. A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides (the components of DNA). VNTR's are 200 base pairs (pairs of nucleotides) long per repeat and a person can have anywhere from 50 to several hundred repeats. This repeat length is inherited. This DNA typing approach was first discovered by the British geneticist Alec Jeffreys in 1985 and is the principle behind today's DNA profiling systems.
The advantage of using a RFLP-based analysis for DNA profiling is that VNTR regions are highly variable in copy number from person to person. Therefore, it is highly unlikely that DNA profiles from unrelated individuals would be identical. However, there are also several drawbacks to this technique. Since these regions are large, it is often difficult to clearly separate the fragment using electrophoresis, which is a technique that uses a DNA sample loaded into a gel that migrates towards a positively charge electric field based on size. For example, larger fragments migrate slower than smaller fragments. This is problematic when the migration of one VNTR is indistinguishable from another VNTR, even if they differ in length. This is due to limited resolution of the gel matrix (only large differences can be detected). A larger amount of DNA (20 nanograms) of purified, high quality DNA is also required for this technique. Thus, DNA samples extracted from crime scene specimens may be not suitable in quality for this type of analysis. High purity in terms of DNA extractions can be compromised according to the source of the sample. If, for example, the sample is blood and is extracted from clothing, the dye from the cloth might alter the mobility of the extracted DNA in the gel, making the analysis difficult.
VNTR analysis has been replaced by Short Tandem Repeat (STR) analysis. STR regions are comprised of 2 base pair repeats that are repeated between 5 to 15 times. STR analysis is currently the standard approach to forensic DNA profiling. This is mainly because shorter repeat sequences are easier to analyze.
STR analysis is faster, less labor intensive, and can be automated. A single reaction can analyze 4 STR regions using very little DNA (only one nanogram is usually sufficient). If only a small amount of DNA is recovered or if it is degraded, it may be possible to use STR analysis, but not VNTR analysis.
Additionally, in VNTR analysis, genomic DNA is digested with restriction enzymes and then run on a gel. The fragments produced are transferred to a membrane and probed with a radiolabeled sequence of DNA that matches the VNTR sequence. The migration of the VNTR fragment on the gel determines their size and generates a pattern. The radiolabeled probe produces dark bands on x-ray film when exposed in a time-dependent and dose-dependant manner. Unlike VNTR analysis, STR analysis uses the polymerase chain reaction to amplify DNA in the region where the STR is located. These PCR products can then be run on a gel in the same manner as the VNTR fragment and using sophisticated computer software with laser controlled equipment, the migration of the PCR products can be compared to control DNA molecules that have a known size. If run together, the size of the unknown STR can be estimated. In this case, STRs are visualized by adding a DNA intercalator such as ethidium bromide into the gel, which intercalates into the DNA and fluoresces (emits) ultraviolet light.
STR analysis, however, is not without its drawbacks, as well. If very little DNA is recovered from a crime scene and it is degraded, not all regions in the genome will amplify, or there will be discriminatory amplification of DNA in only one chromosomal STR region, rather than both. This can significantly affect the results and lead forensic scientists to draw incorrect conclusions. Additionally, there may be substances in the sample that inhibit the PCR reaction. For these reasons, forensics scientists must use a standardized approach that is reproducible and includes all the necessary positive and negative controls for DNA profiling to be used as evidence during a court proceeding.
A significant problem in using DNA profiling as evidence in court proceedings is the possibility that a mistake was made in the sample extraction, preparation, or analysis. For this reason, investigators take precautions to reduce human error. Each forensics laboratory must maintain a high level of quality control and quality assurance standards to prevent this from happening. State and local mandates are being established to standardize these techniques.
Every cell, tissue, or organ in a person's body contains the same DNA pattern, so the United States law enforcement and armed forces has developed databases to collect information related to an individual's DNA identity. This information will be used for identification purposes in missing person cases or to identify the remains of deceased individuals. Other techniques previously used to identify individuals such as using dental records, dog tags, or blood typing have been superceded by DNA profiling, which provides more information and is more conclusive. For example, if two samples have the same blood type, it still is not clear that they came from the same person. Even dental records might not be helpful in cases where the integrity of the sample is compromised to a degree that makes it difficult to match it appropriately. In DNA profiling, even if the deceased person was significantly disfigured, it would still be possible to analyze the sample.
SEE ALSO DNA databanks; DNA evidence, social issues; DNA fingerprint; DNA sequences, unique; DNA typing systems; Mitochondrial DNA typing.
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