More articles by Richard Saferstein, Ph.D. What Is DNA? (Part 1)

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[This article is the second of two parts. Click for Part 1.]

Mitochondrial DNA

Typically, when one describes DNA in the context of a criminal investigation, it's assumed that the subject of attention is the DNA found within the nucleus of a cell. Actually, a human cell contains two types of DNA - nuclear and mitochondrial. The first constitutes the 23 pair of chromosomes contained within the nuclei of our cells. Each parent contributes to the genetic makeup of these chromosomes. On the other hand, mitochondrial DNA (mtDNA) is found outside the nucleus and is inherited solely from the mother.

Mitochondria are cell structures found in all our cells. They are the power plants of our body, providing about 90% of the energy that our body needs to function. What's important from the forensic science perspective is that a single mitochondrion contains several loops of DNA all of which are involved in energy generation. Further, since each cell in our body contains hundreds to thousands of mitochondria, this effectively means that there are hundreds to thousands of mtDNA copies in a human cell. This compares to just two copies of nuclear DNA located in that same cell. Thus, forensic scientists are offered enhanced sensitivity and the opportunity to characterize mtDNA in situations where nuclear DNA is significantly degraded, such as in charred remains, or may be present in small quantity (i.e., hair shaft). Interestingly, in situations where authorities cannot obtain a reference sample from an individual who may be long deceased or missing, a mtDNA reference sample can be obtained from any maternally related relative. However, all individuals of the same maternal lineage will be indistinguishable by mtDNA analysis.

While mtDNA analysis is significantly more sensitive than nuclear DNA profiling, it must be noted that forensic analysis of mtDNA is more rigorous, time consuming, and costly when compared to nuclear DNA profiling. For this reason, at this time, only a handful of public and private forensic laboratories receive evidence for this type of determination.

As was previously discussed, nuclear DNA is composed of a continuous linear strand of nucleotides (A,T,G,C's). On the other hand, mtDNA is constructed in a circular or loop configuration. Each loop contains a sufficient number (approximately 16,569) of A,T,G, and C's to comprise 37 genes involved in mitochondrial energy generation. Two regions of mtDNA have been found to be highly variable in the human population. These two regions have been designated Hypervariable Region I (HV1) and Hypervariable Region II (HV2). As indicated above, the process for analyzing HV1 and HV2 is tedious. It involves generating many copies of these DNA regions by PCR and then determing the order of the ATGC bases constituting the hypervariable regions. This process is known as sequencing. The FBI laboratory, the Armed Forces DNA Identification Laboratory, and other laboratories have collaborated to compile a mtDNA population database containing the base sequences from Hypervariable Regions l and ll.

Once the sequences of the hypervariable regions from a case sample is obtained, most laboratories will simply report out the number of times these sequences appear in the mtDNA database. Currently, however, the mtDNA data base is too small to allow for reasonable estimates of the frequency of occurrence of mtDNA sequences in the population. However, even under the best of circumstances, mtDNA typing will not approach STR analysis in it's discrimination power. Thus, mtDNA analysis is best reserved for samples for which nuclear DNA typing is simply not possible.

Hair DNA Typing The current approach for the examination of hair specimens still includes an examination of morphological characteristics. However, recent major breakthroughs in nuclear DNA (deoxyribonucleic acid) typing has extended this technology to the individualization of human hair. Forensic hair examiners can link human hair to a particular individual by characterizing the nuclear DNA present in the hair root or in follicular tissue adhering to the root When pulled from the head, many hairs will be found with a follicular tag-- a translucent piece of tissue surrounding the hair's shaft near the root. This has proven to be the richest source of DNA associated with hair.

In 1996, the FBI initiated a program to compare human head and pubic hairs through mitochondrial DNA. Importantly, there are many more copies of mitochondrial DNA located in our cells as compared to nuclear DNA. For this reason, the success rate of finding and typing mitochondrial DNA is much greater from samples, such as hair, which have limited quantities of nuclear DNA.

THE COLLECTION AND PRESERVATION OF BIOLOGICAL EVIDENCE FOR DNA ANALYSIS

Since the early 1990s, the advent of DNA profiling has vaulted biological crime scene evidence to a stature of importance that is only eclipsed by the fingerprint. In fact, the high sensitivity of DNA determinations has even changed the way police investigators define biological evidence. In the past, crime laboratories were usually able to extract some useful information from a blood or semen stain, or from a hair deposited at the crime scene. Today, the sensitivity of PCR means that 1 nanogram (one billionth of a gram) or less of DNA can yield sufficient information to individualize evidence. With this technology in-hand, the horizon of the criminal investigator extends beyond the traditional dried blood or semen stain to include stamps and envelopes licked with saliva, a cup that has come in-contact with a person's lips, or a bedsheet containing dead skin cells. Table II illustrates the power of DNA as a creator of physical evidence.

The evidence collector must handle all body fluids and biologically stained materials with a minimum amount of personal contact. All body fluids must be assumed to be infectious; hence, wearing disposable latex gloves while handling the evidence is required. Latex gloves will also significantly reduce the possibility that the evidence collector will contaminate the evidence. These gloves should be changed frequently during the evidence collection phase of the investigation. Safety considerations and the avoidance of contamination also call for the wearing of face masks, shoe covers, and possibly coveralls.

Blood has great evidential value when a transfer between a victim and suspect can be demonstrated. For this reason, all clothing from both victim and suspect should be collected and sent to the laboratory for examination. This procedure must be followed even when the presence of blood on a garment does not appear obvious to the investigator. Laboratory search procedures are far more revealing and sensitive than any that can be conducted at the crime scene. In addition, blood should also be searched for in the less than obvious places. For example, the criminal may have wiped his or her hands on materials not readily apparent to the investigator. Efforts must be made to find towels, handkerchiefs, or rags that may have been used and then hidden. Attention should be given to examining floor cracks or other crevices that may have trapped a quantity of blood.

TABLE 3

Source: National Institute of Justice- U.S. Dept. of Justice

The packaging of biological evidence in plastic or airtight containers must always be avoided, because the accumulation of residual moisture could contribute to the growth of DNA-destroying bacteria and fungi. Each stained article should be packaged separately in a paper bag or in a well-ventilated box. If feasible, the entire stained article should be packaged and submitted for examination. If this is not possible, the dried blood is removed from a surface with the aid of a sterile cotton-tipped swab lightly moistened with distilled water from a dropper bottle. A portion of the unstained surface material near the recovered stain must likewise be removed and placed in a separate package. This is known as a substrate control. The forensic examiner might use the substrate swab as a control to confirm that the results of the tests performed were brought about by the stain and not by the material on which it was deposited. However, this practice is normally not necessary when DNA determinations are carried out in the laboratory. One point is critical, and that is the collected swabs must not be packaged in a wet-state.

All packages containing biological evidence should be refrigerated or stored in a cool location out of direct sunlight until delivery to the laboratory. However, one common exception is blood mixed with soil. Microbes present in soil will rapidly degrade DNA. Therefore, blood in soil must be stored in a clean glass or plastic container and immediately frozen.

Biological evidence will attain its full forensic value only when an analyst can compare each of its DNA types to known DNA samples collected from victims and suspects. For this purpose, at least 7 cc of whole blood should be drawn from individuals by a qualified medical person. The blood sample should be collected in a sterile vacuum tube containing the anticoagulant EDTA (ethylenediamine tetraacetic acid). In addition to serving as an anticoagulant, EDTA inhibits the activity of enzymes that act to degrade DNA. Prior to delivery to the laboratory, the tubes must be kept refrigerated (do not freeze) while awaiting transportation to the laboratory. Besides blood, there are other options for obtaining control DNA specimens. The least intrusive DNA control and one that can readily be used by non-medical personnel is the buccal swab. Here, cotton swabs are placed in the subject's mouth and the inside of the cheek is vigorously swabbed, resulting in the transfer of buccal cells onto the swab.

If an individual is not available to give a DNA control sample, there are some interesting alternatives available to evidence collectors to include: a toothbrush, combs and hair brushes, a razor, and earplugs.

ISSUES INVOLVING CONTAMINATION

We have previously seen forensic scientists are capable of detecting extremely small quantities of DNA from biological evidence. With increased sensitivity comes a greater chance that accidental contamination can be detected in crime scene evidence. Contamination can occur by introducing foreign DNA into a stain while collecting it; or there can be a transfer of DNA when items of evidence are in contact with each other.

Fortunately, an examination of DNA band patterns in the laboratory readily reveals the presence of contamination. For example with an STR, one will expect to see a two band pattern. If one observes more than two bands, it becomes apparent that one could be dealing with a mixture of DNA from more than one source.

There are some relatively simple steps that crime scene investigators can take in order to minimize the possible occurrence of contamination of biological evidence:

1. Always wear disposable latex gloves when collecting biological evidence;
2. Always collect a substrate control for possible subsequent laboratory examination;
3. Pick up small items of evidence such as cigarette butts and stamps with clean forceps. Disposable forceps are to be used so that they can be discarded after a single evidence collection; and
4. Always package each item of evidence in its own well-ventilated container.