Southern Blots

As DNA analysis methods were being developed, it was common to separate DNA fragments of different sizes on agarose gels, as described in the preceding section. Many times, though, scientists wanted to do further testing steps that just would not work in gels. A biochemist named Edwin Southern came up with a method to solve this problem in 1975.

Southern’s method employs a membrane made of nitrocellulose or nylon, which is laid onto the gel after the DNA fragments have been separated. The gel and membrane are then placed into a buffer solution (a solution that resists change in pH), and some absorbent material is placed on top of the membrane. The absorbent material draws the solution upward, through the gel and through the membrane, but the DNA fragments in the gel are too big to go through the membrane, so they stick to it. Because the membrane is the same size as the gel, it retains the original position and orientation of the DNA fragments in the gel.

Once the DNA fragments are on the membrane, they are stable and cannot easily be removed. Furthermore, the membrane is tough and resilient, allowing scientists to do further tests on the DNA right on the membrane. This process of transferring DNA fragments from a gel to a membrane is called “Southern blotting,” and the resulting membrane is often just called a “blot.” This procedure was an important part of the earliest kind of forensic DNA typing, and it is still used regularly in research labs.

Gel Methods

Scientists often need to see whether DNA was successfully prepared from cells or tissues in evidence, how big the molecules are, and how much DNA is in the preparation. Gel methods are commonly used for these purposes. Gel methods provide a way of visualizing DNA.

DNA is a very large molecule that is highly negatively charged. As a result it can be moved by electrophoresis in a gel medium. Electrophoresis is a procedure that uses an electronic field to move big, charged molecules. Agarose gels (similar in consistency to Jell-O) provide a good medium for DNA movement in an electric field. An electrophoresis setup consists of the agarose gel placed in a chamber that separates a positive from a negative compartment and is filled with a solution containing charged particles. The diagram on page 33 shows an electrophoresis setup. A power supply sets up a current in the gel. DNA specimens placed in wells near the negative end will migrate toward the positive end, because DNA is very negatively charged and because unlike charges attract.

Different sized DNA molecules travel different speeds and distances. The larger the DNA molecule, the more slowly it migrates. Thus, the distance a DNA molecule travels gives an indication of how big it is. To help in these estimates scientists typically add standards and calibrators (proteins of known size) to the gel and run them along with the specimens. One factor affecting the size of DNA fragments is degradation. Environmental exposure can cause the DNA in a forensic specimen to degrade, to break into smaller fragments. As a result the specimen will contain an array of different sized DNA molecules. Whereas intact, undegraded DNA appears as a tight band on the agarose gel, degraded DNA is visible as a streak.

DNA is not, however, visible by itself, in a test tube, or on a gel. One must add another material to the DNA to make it visible, and certain dyes that can tuck themselves into the folds of the double helix are used for this purpose. When the DNA-dye complex is illuminated by ultraviolet (UV) light, the DNA fluoresces, so it can be seen. The gels themselves change and degrade over time, so they cannot be kept and stored. Consequently, lab scientists regularly take pictures of these UV illuminated gels to make a permanent record of the outcome of electrophoresis. Furthermore, photography can actually improve visibility of the results. The fluorescent DNA appears white on an otherwise black background in the photo, and it is sometimes easier to see things in the picture than on the gel itself.

Mitochondrial DNA Inheritance

Because mitochondria have their own DNA, separate from nuclear DNA, one can speak of a “mitochondrial genome,” all the DNA contained in a mitochondrion. Cells have many mitochondria, so there are multiple copies of mtDNA in every cell. The MtDNA genome is significantly smaller than the nuclear genome. The nucleus has about 3.5 billion base pairs in its DNA; the mitochondrion has about 16,500 base pairs in its DNA.

All the variability between people that is forensically useful can essentially be seen by looking through a couple of short sequences in the hypervariable regions. In practice, forensic scientists copy the sequences of interest using the polymerase chain reaction and then analyze the copies. Unlike nuclear DNA typing, which consists of determining the sizes of tandem-repeat regions, mtDNA analysis is sequencing. The variability from person to person in mtDNA consists of a few variations in the base sequence. Thus, the PCR products (the copies) from the hypervariable regions have to be sequenced (the order of the bases in the DNA strand has to be determined).

Mitochondria and Mitochondrial DNA

The DNA discussed so far is found in the nucleus of the cell, in the chromosomes. In addition to this nuclear DNA, cells also contain DNA in small structures outside the nucleus called “mitochondria.” The accompanying diagram of a generalized animal cell shows the nucleus and mitochondria, as well as the other cell structures. Mitochondria contain the cell’s energy-processing machinery, but they also contain a small amount of DNA. Mitochondrial DNA (mtDNA) is inherited entirely from one’s mother. There is no paternal contribution.

Mitochondrial DNA, unlike its nuclear counterpart, is circular. In that respect it is similar to some bacterial genomes, which consist of a single circular DNA molecule. It is now known that over long periods of time mtDNA occasionally undergoes mutations and that these mutations are stable and passed along from mother to offspring. Anthropologists, scientists who study the variation and evolution of human beings, use mtDNA to follow patterns of human migration over time. Some of the mtDNA codes for specific proteins, but mtDNA also has a region (called the “control region,” or “D-loop”) that, like some sections of nuclear DNA, is subject to a great deal of polymorphism. It can be divided into two hypervariable regions, designated HV1 and HV2, which are 342 and 268 base pairs (bp) in length, respectively. Hypervariable means that these regions are especially prone to random mutations over time, and most of the variability in mtDNA from person to person is found here.

Forensic mtDNA typing is sometimes used in cases when nuclear DNA typing fails or cannot be done. It is also used in trying to identify human remains. Mitochondrial DNA is quite robust in some tissues, especially older or weathered specimens such as old bones. That is the basis for its use in the identification of skeletal remains, which include no soft tissues (and hence no nuclear DNA). In addition, hair shafts contain mtDNA but no nuclear DNA. (Hair roots have nuclear DNA, but many hairs found as evidence have no roots. They are shed from the human body.) So, any DNA analysis on hair shafts must be mtDNA typing.

The DNA Variation of Interest to Forensic Scientists

The regions of DNA that forensic scientists use to individualize people contain repeated sequences. There are different types of repeat-sequence DNA. A repeated sequence may be found in many different places in the genome. These can be called “interspersed” sequences. Some repeated sequences are head-to-tail repeats of a sequence altogether at one location within the DNA. These are sometimes called “tandem” repeats, and they are the ones that forensic scientists use.

In the context of these regions of DNA the variation between people consists of the number of repeats at a tandem-repeat location. One person might have 10 repeats, and someone else might have 12 or 14 or some other number. The physical structures that contains the DNA are the chromosomes, and humans have 46 chromosomes, which are grouped into 23 pairs. One member of each pair is inherited from one’s mother, and the other comes from one’s father. Thus, everyone has a pair of these tandem-repeat regions. And there may be a different number of repeats on one chromosome compared with the other. Analyzing a person’s DNA for several different DNA locations that have tandem repeats can reveal the high degree of individuality that it represents.

Structural Variation in DNA among Different People

It is often remarked that no two people except identical twins have the same DNA. As far as scientists now know, the statement is true, but like many generalizations, it hides a lot of the detail. What the human genome project has shown is that about 20 percent of human DNA actually specifies protein structure. Much of the DNA that specifies protein structure is pretty similar among different people. The differences that do exist—polymorphism in DNA—cause protein and enzyme polymorphism.

Polymorphism in DNA is simply a base change here or there from one person to another. A single base change in coding DNA might cause a different amino acid to be inserted into the protein; but some single base changes would not even do that, because several triplet sequences of the genetic code can specify the same amino acid. As long as the base changes do not cause too great a change in the protein structure, the protein is still functional. These single-base changes in DNA over time become mutations, and they occur in all cells. Mutations that cause major disruption in the structure (and thus function) of vital proteins generally do not allow the organism that has them to survive, meaning that the mutation does not survive either. But many mutations do survive and create the polymorphism in DNA and proteins that is so common.

In spite of many single-base differences throughout the genome, there is considerable similarity in the DNA of most people. It is not realistic right now to sequence large segments of DNA just to find the differences between people because the process would be too burdensome. The Human Genome Project has provided considerable information about where the differences are, however, and technology is rapidly being developed that will allow searches for hundreds or thousands of small differences all at once.

There is another kind of variation in DNA, however, that has been exploited for forensic DNA typing. As already noted, about 20 percent of human DNA specifies protein structure. What about the remaining 80 percent? What does it do? No one is sure, but there is something very interesting about much of this remaining “nonfunctional” DNA: It has a lot of repeated sequences.

DNA Is the Genetic Material

In 1944 Oswald T. Avery, Colin M. MacLeod, and Maclyn McCarty of the Rockefeller Institute in New York published a paper in the Journal of Experimental Medicine that was to become a classic. Scientists had suspected for some time that DNA was the genetic material, but it had not been experimentally established unequivocally. The question was, Is it really DNA that carries information from one generation to the next?

There are two forms of the pneumococcus bacterium, called R and S. R is a nonencapsulated and nonvirulent form, while S is an encapsulated, virulent (infectious) form. It was known that the R form could be transformed into the S form in an animal by injecting a heat-killed preparation of S and a small quantity of living R. Somehow the R form that does not cause disease was being transformed into the S form that causes pneumonia. Avery, MacLeod, and McCarty wanted to find out the nature of the “transforming” principle. Was it DNA, or was it protein? The question is not as simple as it sounds, because DNA in the nucleus of cells has some proteins associated with it.

They purified the “transforming” principle, the chemical material that they could show was bringing about the R to S transformation, and tested it using a number of methods to help characterize its nature. The methods included chemical analysis, enzyme digestion, and serological reactions. Chemical analysis indicated that the material was consistent with the known composition of DNA but not of protein. Using different enzymes to digest the material (in order to try to destroy the transforming activity), the researchers showed that enzymes that disrupt DNA disrupted the transforming activity. Finally, the serological reaction testing showed that the cell’s non-nuclear material was not involved in the transformation.

The results of the experiments showed convincingly that DNA was responsible for determining whether the cell was R or S. The DNA was dictating the cell type and thus its disease-causing ability. Thus, DNA was the genetic material, the material that controlled cell function. By extension DNA was thus the material that carries the information for dictating cell function from generation to generation, because DNA is what passes from generation to generation. Many other experiments by many scientists have confirmed these findings.

The Denise Johnson Case

In May 1992 the body of a woman later identified as Denise Johnson was found outdoors in the brush near some paloverde trees in Maricopa County, Arizona. Her clothing was scattered about the area, and she had been bound with cloth and braided wire. She was from nearby Phoenix and appeared to have been murdered and left at the location recently. A pager recovered at the scene led the police to a suspect, a man named Mark Bogan. The investigation developed circumstantial, but not definitive, evidence against him. One of the paloverde trees at the scene appeared to have been damaged, possibly by a vehicle. A search of the suspect’s pickup, pursuant to a warrant, revealed seed pods from a paloverde tree in the truck bed.

The suspect admitted that he had picked up Johnson, who had been hitchhiking, and had sexual relations with her in the pickup. But he said he had made her get out of the truck after they had argued. He denied being at the crime scene, and he denied killing her.

Police obtained the assistance of a plant molecular genetics specialist, Dr. Timothy Helentjaris of the University of Arizona, who could compare the DNA profile of the seed pods recovered from the suspect’s pickup with those of the trees in the vicinity of the crime scene. The geneticist conducted blind tests on a number of paloverde trees, and the tests showed that each exhibited a different profile. The seed pods from the pickup truck showed identical profiles (indicating that they fell from the same tree), and their profile matched that of one particular tree at the scene. This evidence went a long way toward convincing the trial jury that the suspect’s pickup was indeed at the crime scene, a fact that he had denied.

The plant genetics expert in this case was a university professor. He used a DNA profiling technique called RAPD (randomly amplified polymorphic DNA) that is not regularly used in forensic labs but is common in research. It is a good technique for looking at genetic variation in organisms whose total genetic makeup, or genomes, have not been mapped or sequenced very thoroughly (as was the case with the paloverde trees). The court allowed the evidence because the expert did a good job of running his tests blind and of establishing that there was much detectable variation in the trees.

Blood Patterns

The interpretation of blood patterns is the reconstruction aspect of forensic blood analysis. It is distinct from identification, species testing, and DNA analysis. During violent events blood can drip from a source, or it can be spattered onto floors, walls, and objects. The patterns the blood forms on the surfaces can help a forensic scientist know what type of event caused the blood to spatter in the first place, how much energy was involved, and possibly something about the direction of a moving source and the angles at which blood droplets hit the surfaces.

Blood patterns at crime scenes are important sources of information about the events that took place there. If there is a possibility that more than one person was bleeding, the laboratory analyzes specimens of the dried blood, first, to make sure it is blood; then, to make sure it is human; and finally, to analyze its DNA profile. Blood patterns will have different meanings if there is more than one blood source. As a result the identification and individualization steps in blood analysis always precede the reconstruction step.

Identifying Human Remains

Most societies consider it important to recognize the death of an individual and to dispose of his or her remains in a particular way. Most people die under circumstances where their identity is not in question. But in cases where deaths are sudden or unexpected, occur without any medical oversight (outside a clinic or hospital), or might involve foul play, they are investigated by medical examiners or coroners. The first step is identifying the body. Identification of remains is also one of the major goals in mass disaster situations, such as airplane crashes. The U.S. Armed Forces are likewise dedicated to identifying the remains of military personnel who die in the line of duty.

Parentage Testing

Not too long after the discovery of blood types, it became clear that they were inherited. The ability to test for discrete, inherited characteristics opened the way to using genetic testing as a means of trying to establish (or disprove) parentage.

A child’s mother is usually known because there is some record of the child being born to her, so most disputed parentage cases involve disputed paternity. Most of these cases are brought in family courts and attempt to establish paternity in order to give the court a basis for ordering a man to pay support for his child. Today, with DNA-typing methods the chances of falsely including a true nonfather are exceedingly small; in other words, if DNA testing results provide a high probability that a particular man is a child’s father, then he almost certainly is. For the family courts a DNA parentage inclusion is equivalent to proof of parentage. By the same token DNA typing will virtually always exclude a true nonfather.

Methods Used to Manipulate and Analyze DNA

Forensic DNA typing was made possible not only by advances in knowledge about DNA structure and function but also by advances in the methods and techniques allowing DNA to be manipulated. Different manipulation methods apply to different DNA-typing technologies. For example, nuclear DNA was first typed using the restriction fragment length polymorphism (RFLP) technique. This procedure did not rely on the polymerase chain reaction but on restriction enzymes and Southern blots. (Southern blotting is discussed later in this chapter.) By contrast, the current typing procedure does rely on PCR but does not make use of restriction enzymes.

DNA Analysis Meets Forensic Science

In the 1980s DNA scientists (molecular geneticists) focused on the repeat-sequence polymorphism within DNA. For most researchers these repeat-sequence regions were “road signs” along the sequence of letters (bases) as different laboratories worked on sequencing the entire human DNA. Dr. Alec Jeffreys at the University of Leicester in the United Kingdom realized that these polymorphisms provided excellent tools for human identification in affiliation cases, especially when many regions were examined simultaneously. He called these patterns “DNA fingerprints,” a term that has stuck, especially in the popular media. Most forensic scientists dislike the term because it can create confusion between DNA and conventional fingerprints, and because there are some differences between DNA individuality and fingerprint individuality. Jeffreys published several papers on this subject in the prestigious scientific journal Nature in 1985.

Around this same time, in 1983 and in 1986, two teenage girls had been raped and murdered in the small village of Narborough in Leicestershire, England (see sidebar “The Narborough Murders”). Jeffreys and DNA technology would be drawn into this case, and its outcome became the flash point for the development of DNA-typing methods in forensic science laboratories worldwide.

Within a couple of years forensic science laboratories all over the world had acquired the tools to perform the new DNA-typing technique. Jeffreys’s name will be forever linked with this revolution. In 1998, at its 50th anniversary meeting in San Francisco, the American Academy of Forensic Sciences paid special tribute to Jeffreys in recognition of his contributions.

The DNA Era

Much of the repeated sequence data in the human genome is not functional; it does not specify protein structure. It has been called “junk DNA,” though it may have functions that are not yet clear to scientists. About 20 percent of human DNA is functional, in the sense that it codes for protein. And, within that 20 percent, there is considerable similarity in sequence among different people. This is exactly what would be expected; the structure of functional DNA would be conserved. Any major alterations in the sequence of the functional DNA would lead to problems with the specification of protein structure and would likely cause problems for the individual.

Changes in one or a few bases in DNA are called mutations, and they do occur. Study of variations in functional proteins, such as hemoglobin, makes it clear that some mutations are innocuous. Scientists know this because these mutated versions of hemoglobin are found in living people. In other words, there has been a mutation in DNA, and it has caused a change in the hemoglobin protein, but the person with the mutation is alive and well. The mutation did not therefore affect the functionality of the hemoglobin. But many mutations do cause problems with protein functionality. These problems often lead to serious medical problems. Many mutations probably lead to early death—so early in the development of an embryo that the mother may not yet even realize she is pregnant. Scientists will never see the mutations that prevent such an embryo from surviving.

In the 80 percent or so of DNA that does not specify protein structure, there is enormous sequence and repeat-sequence polymorphism. For the most part it does not seem to have any negative consequences for the individual and can be exploited for purposes of identification, as has been done by forensic scientists.

Analysis and DNA Typing of Blood and Other Physiological Fluids

Once established as human, blood and physiological fluid stains and traces are next DNA typed to find out who may have deposited them. DNA profiling means finding out the DNA types at several different genetic loci.

Genetic loci, or different locations on the DNA, are explained later in the book. The combined set of types at the different locations is the profile. It is the profile that is actually characteristic of an individual.