DNA Fingerprint (World of Forensic Science)
DNA (deoxyribonucleic acid) represents the blueprint of the human genetic makeup. It exists in virtually every cell of the human body and differs in its sequence of nucleotides (molecules that make up DNA, also abbreviated by letters, A, T, G, C; or, adenine, thymine, guanine, and cytosine, respectively). The human genome is made up of 3 billion nucleotides, which are 99.9% identical from one person to the next. The 0.1% variation, therefore, can be used to distinguish one individual from another. It is this difference that can be used by forensic scientists to match specimens of blood, tissue, or hair follicles to an individual with a high level of certainty.
The complete DNA of each individual is unique, with the exception of identical twins. A DNA fingerprint, therefore, is a DNA pattern that has a unique sequence such that it can be distinguished from the DNA patterns of other individuals. DNA fingerprinting is also called DNA typing.
DNA fingerprinting was first used for sample identification after the geneticist Alec J. Jeffreys from the University of Leicester in Great Britain discovered that there are patterns of genetic material that are unique to almost every individual. He called these repetitive DNA sequences "minisatellites." The two major uses for the information provided by DNA-fingerprinting analysis are for personal identification and for the determination of paternity.
DNA fingerprinting is based on DNA analyzed from regions in the genome that separate genes called introns. Introns are regions within a gene that are not part of the protein the gene encodes. They are spliced out during processing of the messenger RNA, which is an intermediate molecule that allows DNA to encode protein. This is in contrast to DNA analysis looking for disease causing mutations, where the majority of mutations involve regions in the genes that code for protein called exons. DNA fingerprinting usually involves introns because exons are much more conserved and therefore, have less variability in their sequence.
DNA fingerprinting was originally used to identify genetic diseases by linking disease genes within a family based on the inheritance of the segregating markers and the likelihood that they would be in close proximity, but it also became used for criminal investigations and forensic science. In general, the United States courts accept the reliability of DNA analysis and have included these results into evidence in many court cases. However, the accuracy of the results, the cost of testing, and the misuse of the technique have made it controversial.
In forensics laboratories, DNA can be analyzed from a variety of human samples including blood, semen, saliva, urine, hair, buccal (cheek cells), tissues, or bones. DNA can be extracted from these samples and analyzed in a lab and results from these studies are compared to DNA analyzed from known samples. DNA extracted from a sample obtained from a crime scene then can be compared and possibly matched with DNA extracted from the victim or suspect.
DNA can be extracted from two different sources within the cell. DNA found in the nucleus of the cell, also called nuclear DNA (nDNA) is larger and contains all the information that makes us who we are. It is tightly wound into structures called chromosomes. DNA can also be found in an organelle within the cell called the mitochondria, which functions to produce energy that drives all the cellular processes necessary for life. Mitochondrial DNA (mtDNA) is much smaller, contains only 16,569 nucleotide bases (compared with nDNA, which contains 3.9 billion) and it is not wound up into chromosomes. Instead, it is circular and there are many copies of it.
Nuclear DNA is analyzed in evidence containing blood, semen, saliva, body tissues, and hair follicles. DNA from the mitochondria, however, is usually analyzed in evidence containing hair fragments, bones, and teeth. Mitochondrial DNA analysis is typically performed in cases where there is an insufficient amount of sample, the nDNA is uninformative, or if supplemental information is necessary.
Unlike nDNA, where one copy of a chromosome comes from the father and the other from the mother, mtDNA is exclusively inherited from the maternal side. Therefore, the maternal mtDNA should be the same as her offspring. This can be helpful in cases where it is not possible to obtain a sample from the suspect but it is possible to obtain a sample from one of the suspect's biologically related family members. By doing so, the suspect can be excluded as the culprit of a crime if the results indicate that the relevant family member's mtDNA does not match the mtDNA fingerprint from the sample.
Mitochondrial DNA can be informative in a different way than nDNA. Less than 10% of the mitochondrial genome is noncoding and localized in a region called the D-loop. In this region, there are sequence variations that are inherited that can be used for forensic purposes. These regions, called hypervariable regions, are broken down into two sections: HV1 and HV2. It is within these regions that inherited sequence variations can be identified.
One of the main reasons mtDNA analysis can be helpful to forensic scientists is that in some tissues, mitochondrial DNA is in excess compared to nDNA. As nDNA exists in chromosomes and there are only two copies of each chromosome (one inherited maternally, the other paternally) per cell, the nDNA copy number is much smaller. The mitochondrial genome can have a copy number of 20 per organelle and in some cases the number of organelles can reach the hundreds. For example, in muscle tissue, where the demand for energy is highest, there are a larger number of copies of the mitochondrial genome. Analysis of mtDNA, therefore, can be particularly helpful in forensic cases where sample integrity or size is compromised or when confirmation is needed.
There are many methods that forensic scientists use to determine the sample's DNA fingerprint. Once DNA is extracted, it can then be analyzed using a variety of molecular genetics techniques. In some cases, there is not enough DNA to directly evaluate it. If this occurs, a technique called the polymerase chain reaction (PCR) is used to amplify the genomic DNA from a sample. This procedure allows a scientist to amplify a specific sequence of DNA in the genome exponentially, so that it is in large enough quantities to be analyzed.
DNA analysis can be performed by sequencing the amplified DNA fragment using fluorescently labeled nucleotides and a laser that will recognize the nucleotide based on the fluorescent label to which it is attached. This technique is expensive, may not be informative, and is generally not the best approach to DNA fingerprint a sample.
If there is enough DNA, the DNA extracted from the sample can be cut or segmented using specific enzymes (proteins that speed up chemical reactions) called restriction endonucleases that act as molecular scissors by cutting specific sequences that they recognize. By cutting in the same sequence that is present in different locations throughout the genome, a pattern of fragments can be formed. Differences in the sequence patterns between two samples can be due to inherited variations in the DNA that can distinguish two different samples.
Once the DNA is cut, the segments are arranged by size using a process called electrophoresis, whereby an electrical field is generated, pulling the negatively charged DNA toward the positively charged end through a gel-like matrix. The segments are marked with radioactive probes and exposed on x-ray film, where they form a characteristic pattern of black bars. This pattern is called the DNA fingerprint. If the DNA fingerprints produced from two different samples match, the two samples are likely to have come from the same person.
DNA can also be processed and cut with restriction enzymes. If there is a variation in a particular sequence that results in the enzyme no longer recognizing and cutting the DNA (or a loss of the cut site), a larger fragment will be observed when running the DNA in a gel by electrophoresis. Using a chemical that binds to DNA (called ethidium bromide) and fluoresces when it is excited by ultraviolet radiation, the fragments can be observed on a gel based on size. Bigger fragments will migrate more slowly in the gel. An individual with the sequence variation in which the enzyme does not cut would have a longer size fragment than the individual with the variation the enzyme does cut.
The original DNA fingerprinting procedure used Variable Number Tandem Repeats (VNTR), which are repetitive DNA sequences that are spread throughout the genome in noncoding regions. These targets are large, with repeat numbers that are variable from person to person and have a repeat size composed of hundreds of nucleotides which can be repeated a hundred times.
The biggest problem with using the VNTR-fingerprinting approach is that DNA extracted from samples in a crime scene, such as from a dried blood stain, is often broken up into tiny pieces due in most cases to natural DNA-degrading processes. This can make DNA analysis difficult, unless informative fragments remain intact. Additionally, the smaller the sample, the more likely it will be degraded. For example, a plucked hair might contain up to 30 nanograms (30 ng, or 30 billionths of one gram) of genomic DNA, but a hair shaft without the root might maximally only contain 0.1 ng of DNA. The integrity of the sample as well as the quantity, therefore, can make reliable and definitive identity determination difficult.
More recent approaches have circumvented the problem associated with degraded DNA. Shorter repetitive sequences, or short tandem repeats (STR), were later identified and found to contain repeat core units of three, four, or five nucleotides long and have a complete length of only 8000 nucleotides. Due to the shortness of these sequences, only 50 pg of DNA (which is almost a 1000 times less than that found in a hair shaft without the root) is required. The discriminating power, when analyzing STRs at multiple locations with the genome, can match persons with a probability of 1 in 1015 to a stain. The DNA fingerprint using STR analysis can, therefore, be an extremely powerful technique in forensic sciences.
With the completion of the human genome sequence and the rapid post-genomic characterization of the sequences, it has become easier to analyze samples pertinent for forensic applications. In fact, forensic scientists have been able to link a suspect to the scene of a crime using dried chewing gum, the cells in the saliva from the butt of a cigarette, and cells found underneath fingernails. DNA fingerprinting, therefore, has revolutionized the forensic sciences by its use in investigations and prosecutions of active criminal cases, missing persons investigations, re-examining dead-end cases, post-conviction exoneration, and studies where maternal relatedness is in question.
SEE ALSO Analytical instrumentation; Chemical and biological detection technologies; DNA profiling; DNA recognition instruments; RFLP (restriction fragment length polymorphism).