Tuesday, May 17, 2011

DNA Extraction

Any biological fluid or tissue that contains nucleated cells can be used as a source of DNA. A nucleated cell is any cell that has a nucleus. In blood the white blood cells have nuclei and therefore have DNA, but the red blood cells do not. The sperm cells contain most of the DNA that is in semen, but there are also some epithelial cells, which contain DNA. Much less DNA is obtained from a comparable quantity of semen that lacks sperm cells. In vaginal swabs and in buccal (inner cheek) swabs the epithelial cells have DNA. Urine has some cells in it but not too many. Sometimes enough DNA can be isolated from urine to enable successful typing, but not always.

All specimens containing nucleated cells are handled pretty much the same way in DNA analysis. The specimen is combined with an enzyme called proteinase K, which hydrolyzes the proteins nonspecifically; that is, it catalyzes the breakdown of cellular protein. Proteinase K also digests cell and nuclear membranes, releasing all the contents of the cell and its nucleus into the solution. The result of this process is called a “digest.” Next, it is common to extract DNA from this mixture using a two-phase system of phenol and chloroform. Two-phase means that these two liquids do not mix; when combined, they form two separate layers in a test tube. The two phases and the digested specimen solution are then mixed by shaking, and this causes much of the protein to move into the chloroform layer. The chloroform layer is discarded, while the phenol phase, which contains the DNA, is kept. This extraction process may be repeated two or more times.

The DNA is finally isolated from the phenol phase either by precipitation with ethyl alcohol or filtration using a miniconcentrator. DNA is insoluble in ethyl alcohol, so adding it to a DNA-containing solution causes the DNA to fall out of solution. A miniconcentrator is a small filtration device. The filter retains DNA but allows solutions of phenol, buffer, and so forth to pass through. This device can essentially wash the phenol out of the DNA. The DNA is then recovered in a buffer solution in which it is stable. The miniconcentrator procedure is now common in forensic labs.

There are other ways of processing the proteinase-K digests, such as using special columns lined with materials that adsorb DNA under certain conditions, then release it under other conditions. The mechanism of this adsorption involves oppositely charged molecules attracting one another. A positively charged molecule lining the column will bind DNA. By changing the pH of the buffer solution passing through the column, one can alter the net charges on the column molecules and the DNA so that DNA can be released from the column.

Separation, Labeling, and Detection of DNA Segments

Suppose a scientist were looking for a certain sequence pattern in a mixture of DNA fragments of different sizes. The mixture could be separated by electrophoresis on an agarose gel and then fixed on a membrane by Southern blotting. Then, in order to locate the desired DNA sequence pattern on the blot, a single-stranded segment of DNA called a “probe” could be added directly to the blot.

A probe is a single strand of DNA with a sequence complementary to the target strand of DNA. There are methods to easily label the probe—and thus visualize the target sequence that the probe binds with—using radioactive phosphorus-32 (32P) labels. Once the probe is labeled, it can be placed in a solution with the membrane-containing mixture of all the separated DNA fragments. Under controlled conditions the probe will base pair only with the exactly complementary sequence; this process is called “hybridization.” The reason that the probe can base pair (hybridize) with its complementary sequence directly on the membrane is that the Southern blotting process has already denatured the DNA on the membrane, that is, rendered it single stranded.

The excess probe and solution can then be washed away, leaving the membrane with a radioactive probe adhering to one DNA fragment. The scientist can now visualize the target DNA sequence by placing this membrane against a sheet of X-ray film. The radioactivity exposes the film just as X-rays would. When the film is developed, it shows an image of the DNA fragment that had the radioactive label on it. This process, illustrated in the diagram, is called “autoradiography.” The first forensic DNA typing was done in this way. But radioactive materials such as 32P pose a health hazard, so it is expensive and cumbersome to handle and store them in the safe, legal way. Later, a method using light-emitting (chemiluminescent) probes was developed so scientists could avoid having to work with the radioactive phosphorus.

As already discussed, scientists can separate DNA fragments of different sizes using electrophoresis on agarose gels, but the electrophoresis can also be carried out in tiny capillaries that are filled with a solution of buffer and polymers (somewhat comparable to agarose). This process is capillary electrophoresis (CE) and has some significant advantages over traditional gel electrophoresis. One is that heat does not build up as much in the capillary. Heat buildup is a problem in electrophoresis, both because it can denature protein or DNA molecules and because it can change the properties of the medium (the agarose or buffer solution) in a way that affects the migration properties of the molecules. If heat buildup is not controlled, it can affect the reproducibility of electrophoresis.

 DNA probes and Southern blotting. Probe:
Short sequences of single-stranded DNA, 20-50 nucleotides (building blocks) long, can be chemically synthesized and them made radioactive or fluorescent. This DNA can base pair with a complementary DNA or RNA strand, labeling it as either radioactive or luminescent. Southern blotting:
1. The DNA is fragmented by the addition of a restriction enzyme (biological catalyst that cuts DNA at specific sites).
2. The fragments are next separated, according to their length, into invisible bands on electrophoresis gel.
3. The separated DNA fragments can then be transferred to a nitrocellulose or nylon
membrane. The DNA molecules are made to stick permanently to the membrane.
4. When a radioactive DNA probe solution is added to the membrane and then gently washed off, some of the probes will remain attached to any DNA fragments that have a complementary sequence.
5. The membrane is placed against a photographic film. 6. The film darkens wherever the radioactive DNA probe has bound to the membrane. The probe has identified which DNA fragment contains the complementary DNA sequence for the probe.

Tuesday, April 26, 2011

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.

Monday, April 25, 2011

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.

Sunday, April 24, 2011

Restriction Enzymes

Restriction enzymes were important in the RFLP forensic DNA-typing procedure. Many different bacteria make special enzymes called restriction endonucleases, or restriction enzymes. There are many different restriction enzymes, but they all do one thing: They cut the doublestranded DNA molecule at a specific place. Strictly speaking, the enzyme itself does not “cut” DNA; it catalyzes the hydrolysis of DNA. Hydrolysis means that a molecule is separated into two parts and a molecule of water (H2O) is added in the process. But, as a kind of shorthand, molecular biologists often talk about restriction enzymes “cutting” DNA.

A restriction enzyme cuts DNA at a specific place along the chain because it recognizes a short base sequence of four to seven bases. The restriction enzyme HaeIII, for example, recognizes the sequence . . . GGCC . . . and EcoR1 recognizes the sequence . . . GAATTC. Four additional examples of restriction endonucleases are illustrated in the figure. The heavy arrows in the figure show the cut points in double-stranded DNA at the recognition sequence. The earliest forensic DNA-typing method involved the use of restriction enzymes.

Saturday, April 23, 2011

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).

Friday, April 22, 2011

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.

Thursday, April 21, 2011

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.

Wednesday, April 20, 2011

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.

Tuesday, April 19, 2011

The Polymerase Chain Reaction

The original DNA-typing method, the one Jeffreys used in the Narborough case, was too cumbersome and time consuming to be useful for casework and database construction. A different, faster technology was needed. It was developed in 1985.

The polymerase chain reaction is one of the most stunning breakthroughs in all DNA science. The idea is attributable to Kary Mullis, who worked for Cetus Corporation at the time; he received the Nobel Prize in chemistry in 1993 for his discovery. Cetus scientists commercialized PCR, and it has been extensively used in research and application laboratories, including forensic science labs, all over the world.

Essentially, PCR copies a segment of DNA multiple times using an enzyme called a DNA polymerase. The segment that is to be copied is specified, or defined, by two primers. In this way a small amount of DNA can be used to make a very large number of copies of a desired small portion of the total molecule. This process has been extremely useful in basic and applied biomedical research and as the basis for clinical and forensic tests.

Forensic DNA typing is based on a simple principle: A person’s DNA type is a statement about how many times the head-to-tail repeat sequences are repeated. Chromosomes are paired, and on these pairs one chromosome has a repeat number inherited from the mother, while on the other chromosome is a repeat number inherited from the father. In the majority of people the numbers are different. Combining information about these numbers of repeats at several regions in the DNA can generate a profile that is very unlikely to be found in more than a single individual.

Monday, April 18, 2011

The Narborough Murders

In 1983, in England, a 15-year-old girl who was walking home along a country lane near Enderby became the victim of rape and murder. The investigation yielded no suspects. Three years later another young girl was sexually assaulted and murdered in a similar manner in the nearby village of Narborough. Police arrested a teenager named Rodney Buckland, who worked in a local mental hospital. Buckland had made statements incriminating himself in the second murder but proclaiming his innocence in the earlier one.

The police were certain that Buckland had raped and killed both girls. They sent semen samples recovered from the two victims, along with a blood sample from Buckland, to Alec Jeffreys for examination using his new DNA-typing method. The DNA analysis confirmed that the same offender had committed both crimes as police suspected, but it showed that Buckland was excluded. His incriminating statements were false. Thus, Buckland became the first man ever exonerated by DNA typing. Without it he might well have gone to jail for a very long time.

The police then began a massive manhunt for the true perpetrator. They conducted what might be called a “biological evidence dragnet,” or “DNA dragnet.” All the males in the village between the ages of about 14 and 70 were requested to provide reference blood specimens. Most did so—more than 5,000 in all. At this point DNA typing was very new; forensic science laboratories did not even perform it yet. Jeffreys had agreed to do the DNA typing in his lab at the University of Leicester. Because this early form of DNA typing was very complex and time consuming, the Home Office (U.K. government) forensic science lab first “screened” all the specimens using the conventional genetic-marker systems that were discussed earlier in this chapter. This excluded most of the men as potential semen donors on the basis of their ABO blood types, secretor status, and/or isoenzyme types, and they were thus excluded from further consideration as suspects. The remaining specimens were then sent for DNA typing.

In the first round of DNA typing none of the men’s samples matched the types of the semen donor in the crimes. The perpetrator, a 27-year old named Colin Pitchfork, was ultimately found, arrested, and convicted of the offenses, because he had paid someone to give police a voluntary blood specimen using his name. He bragged about this ruse while drinking in a pub, was overheard, and was turned in to the police. The surrogate, when confronted, admitted to the police what he had done. Pitchfork’s DNA types matched those found in the semen recovered from both victims.

Sunday, April 17, 2011

Development of Forensic DNA Typing

Starting in the 1980s, methods were developed that made DNA sequencing much easier and faster than it had been. These methods could be automated, enabling a very large amount of sequence data to be collected in many laboratories throughout the world. With these methodologies in place in the larger research centers, the U.S. government and a private company made commitments to pursue sequencing the entire human genome. This task, one of the more impressive in the history of biological science, was completed in the late 1990s.

As sequence data accumulated it became clear that a large fraction of human DNA was not involved in specifying protein structure; that is, it did not seem to have any obvious function. It has been called “junk DNA” for this reason. Much, but not all, of this nonfunctional DNA has repetitive sequences. There are several different types of repeat-sequence DNA, but the most important for purposes of human identification, and thus forensic purposes, is a kind that has head-to-tail sequences repeated one after the other along the DNA strand. Many regions in the human DNA have this type of repeated sequence structure, known as tandem repeats. The reason tandem-repeated DNA is useful for human identification is that there is a lot of variation among different people in the number of times the sequence is repeated.

Jeffreys was studying regions of DNA that have the head-to-tail repeat sequences when he developed the first forensic DNA typing. Under certain lab conditions many of these regions can be examined simultaneously using a technique called restriction fragment length polymorphism (RFLP). RFLP will be fully described in chapter 3, but briefly, the technique generates a series of parallel light and dark lines on X-ray film. It looks something like a bar code. Every individual has a different DNA “bar code,” and this observation forms the basis of forensic DNA typing—that is, DNA typing for human identification.

Since the 1980s, when the earliest versions of forensic DNA typing were developed, several new technologies have been developed and implemented. The most important breakthrough was using a technique called polymerase chain reaction (PCR) to make multiple copies of small quantities of DNA in a specimen for subsequent typing.

Saturday, April 16, 2011

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.

Friday, April 15, 2011

Controlling the Synthesis of Proteins

As the Avery-MacLeod-McCarty experiments in the 1940s showed, DNA specifies the cell’s activities. It was now clear that DNA directs the course of cell development and differentiation in multicellular animals, thus forming the individual. It then has a role in maintaining life functions, in repair of damaged cells or tissues, in healing, and in fighting off disease. But scientists did not yet understand how DNA accomplished these activities. Its pivotal role in specifying the sequence of amino acids in proteins was established in the 1960s.

During the decade biochemical methods improved, making it easier to figure out how genes actually worked. The structure of DNA had been established in 1953 by James Watson and Francis Crick, and scientists began unraveling how DNA uses ribonucleic acid, or RNA, to specify the synthesis of proteins. Robert Holley, H. G. Khorana, and Marshall Nirenberg shared the Nobel Prize in 1968 for their key roles in describing this process. They discovered that DNA specifies the structure of RNA and that RNA in turn specifies the structure of protein. DNA works through RNA in specifying protein structure. It does this by way of the genetic code. Three DNA bases in a row specify, through intermediary RNA, a building block of a protein called an amino acid.

The process by which DNA functions to tell the cell how to make its proteins has been called the “central dogma of molecular biology.” Since many proteins are enzymes, whose presence or absence determines whether a certain reaction can occur or not, specification of protein structure is one important way DNA regulates cell activity.

Thursday, April 14, 2011

The Fruit Fly, Bread Mold, and Bacteria Pioneers

Around 1900 the study of genetics really took off. One pioneering group was that of the noted biologist Thomas Hunt Morgan, a faculty member of Columbia University in New York City. Around this time the fruit fly, Drosophila melanogaster, became the geneticist’s research animal of choice. These little flies have many characteristics that are easy to observe under a magnifier or a low-power microscope and that are inherited in simple ways. Fruit flies are fairly easy to maintain in the laboratory, have short reproductive cycles, and produce many offspring. These features are ideal for doing genetic research.

Morgan attracted some very gifted students—A. H. Sturtevant, Calvin Bridges, and H. J. Muller—who would go on to become first-rate geneticists. Morgan was awarded the Nobel Prize in 1933, and Muller received it in 1946, for discoveries in genetics. Along with others these pioneers established the physical basis for inheritance: Genes are carried in structures called chromosomes, of which humans have 46. They confirmed and extended Mendel’s basic rules and also found a number of new genetic phenomena, including the process of crossing over, how mutations can be induced, and the mechanism of sex-linked inheritance.

Molecular genetics is the study of the molecular mechanisms within DNA and RNA that explain the underlying basis of inheritance. One could argue that the era of biochemical genetics, or molecular genetics, began with the studies of George Beadle and E. L. Tatum using the bread mold Neurospora crassa as a research organism beginning in 1941. They were interested in establishing that a gene (within the DNA) was responsible for a specific protein or enzyme, a concept known as the “one gene—one enzyme” hypothesis. The methods to do that kind of detailed biochemical work in the fruit fly were not available at the time, so Beadle and Tatum chose bread mold.

Bread mold has several useful features for genetic research. It can be grown in simple, laboratory-prepared media in petri dishes. Mutations can be induced in the mold that change its nutritional requirements in simple, straightforward ways. This information can then be used to figure out that a gene controlling a particular enzyme in a nutritional pathway has been altered. Their work allowed them to formulate the one gene–one enzyme hypothesis, and in 1958 they, along with Joshua Lederberg, were awarded the Nobel Prize for their discoveries in molecular genetics.

Finally, one of the most famous experiments in the history of biology was conducted by Oswald T. Avery, Colin M. MacLeod, and Maclyn McCarty. In 1944 they showed conclusively that deoxyribonucleic acid, or DNA, is the genetic material. Their cleverly designed protocol, described in the sidebar “DNA Is the Genetic Material,” involved infection of bacterial cells by a virus that normally infected them.

Wednesday, April 13, 2011

Inheritance Among the Pea Plants

The earliest known systematic experiments in genetics were performed by an Augustinian monk named Gregor Mendel (1823–84). Although his work was presented and recorded in a scientific proceeding in 1865, not many people heard about it until more than three decades later. The scientific society and its proceedings, where the work was originally described, were obscure, and no one in mainstream science at the time saw it or paid attention.

Working in the garden of a monastery in Brünn, Austria (modern-day Brno, Czech Republic), Mendel performed carefully controlled experiments on inheritance in common garden pea plants. Mendel studied seven characteristics of the pea plants, including red versus white flower color, tall versus short, and smooth versus wrinkled seed texture.

By crossing plants with known characteristics in controlled breeding experiments and recording the numbers of offspring that had each of the characteristics, Mendel was able to infer how the characteristics were being inherited. He proposed the concept of the gene as the unit of inheritance of these characteristics, and he hypothesized that each individual has a pair of genes for each characteristic, one inherited at random from each parent. His fundamental discoveries are now often  known as Mendel’s laws, and simple inheritance patterns in families are sometimes called “Mendelian.”

Tuesday, April 12, 2011

Using Isoenzyme Types to Include and Exclude Bloodstain Sources

In a suburban community near a large city a woman was violently assaulted in her home. She had arrived home and surprised a burglar. She fought with the intruder briefly and received injuries, from blows and from being cut with a knife. She did not get a very good look at the perpetrator. There had been a series of burglaries in the area, and police had some information from other victims that eventually led them to charge a man with the burglaries and with the assault on this woman.

Police obtained a warrant to search the suspect’s residence for clothes that might have some blood on them from the struggle with the assault victim. They seized a pair of trousers that appeared to be bloodstained. The trousers, along with blood standards from the suspect and the assault victim, were submitted to the forensic laboratory for examination. The stains on the trousers were of human blood, and the genetic typing results were as shown in the following table.







The first column of results is the ABO type of the people and the evidence. The rest of the columns are isoenzyme name abbreviations. Each abbreviation has a specific meaning and a set of characteristics associated with it. But it is not necessary for one to understand all the numbers and letters to understand this case. One can look through the table and quickly see what matches and what does not. The genetic systems are independent, so a nonmatch in any column would be an exclusion.

What the table shows is that the stains on the trousers are not of the suspect’s blood. This fact is clear from the ABO type: He is O, and the stain is A. The stains came from someone else. They could be from the assault victim, because all the system types match. But that does not mean the stains came from her to the exclusion of all other people; this kind of blood analysis is not powerful enough to individualize. So, what can be said or concluded by the analyst?

Here is where population genetics comes into this picture. Population genetics is a big subject, but briefly, scientists can find out how often each different type in each of the systems is seen by typing people in the population for all the genetic systems. These numbers give pretty good estimates of the actual distributions. They are estimates because it is not practical to type everyone; a sample of people is typed. The other important factor is that all these systems are inherited independently; that is, the ABO type that someone inherits from his or her parents does not influence nor is it influenced by the PGM type he or she inherits, and so on. This is like successive flips of a coin, or successive throws of dice. What one gets on the first toss is independent of what one might get on the second toss.

What it all means is that one can multiply the separate occurrences (or probabilities) together to get the combined figure. For instance, the chances of a head on the first coin toss are ½ (or 50 percent). The chances of getting two heads in a row are ½ times ½ (or 25 percent). The chances of getting three heads in a row are ½ times ½ times ½ (or 12.5 percent), and so forth.

The same thing can be done with the data in the table above. It is possible to figure out about how many people in the population would have all the types found on the trouser stains. This figure comes out to about 2 percent for Caucasians (the number would be different if the calculation were done for African Americans, Hispanics, Asians, or another group). So the analyst could tell the court that, if the blood came from a Caucasian, it could have come from about two in every 100 people. In other words, it could have come from the victim in the case, but it could also have come from many other people. In a city of 1 million people, 20,000 would be expected to have the set of types seen in this evidence.

The case shows how several genetic systems can be used together to decrease the size of the population segment that a specimen might have come from. It also shows that even if one gets results that yield a fairly low percentage of the population as possible depositors, there are still quite a few people who share the same types.

Sunday, April 10, 2011

ABO Blood Typing and Exclusion

Let us call the people involved in this case Jane Doe and John Smith. Who they are is not important to understanding the scientific issues. In the 1980s Jane Doe reported a sexual assault in a suburban town adjacent to New York City. The clinical examination she underwent at the time included taking a vaginal swab, among other items, into evidence. Doe was a type B secretor, so she would produce blood group substances B and H. When the swab was analyzed, it showed the presence of blood group substances B and H, as well as semen.

The suspect, Smith, was later apprehended as a result of the police investigation, and Doe identified him as the man who assaulted her. Smith was tested and found to be a type A secretor, so his ABO blood typing was expected to produce blood group substances A and H. No blood group substance A was found in the evidence, however.

What did this finding mean? Did it mean that Smith had to be excluded as a source of the semen? That is almost right. Smith was excluded on the face of things, but there were still a few matters the analyst had to consider.

One such matter is variation in the ratios of blood group substances. People can produce different quantities of these blood group substances in their body fluids. For instance, the relative proportions of blood group components may be different in the same person’s blood and saliva. When a person is tested to find out his or her secretor status, saliva is used. In this case the analyst had to consider the possibility that the A and H quantities could be present in different ratios in the saliva and the semen of the same type A secretor man. It was possible that an A secretor man could have more A in saliva (enough that the lab detected it when the saliva was tested) and less in semen (so that it was not detected in the semen found in the evidence specimen). Under those circumstances the man would still be included as a possible source.

But there is no way of knowing about the A and H levels in saliva versus in semen in any given case. When a person is tested for secretor status, what the lab tests is saliva because this fluid is relatively easy to obtain and getting a saliva specimen from a person is relatively nonintrusive. Forensic scientists are almost never able to test someone’s actual semen, because it is too intrusive for any court to order someone to produce such a specimen. As a result it was generally not possible to sort out these possibilities through ABO blood type testing.

A second matter affecting interpretation of lab results is what the analyst does not know. For instance, if the complainant had another sexual partner besides the person who assaulted her, the analyst would not know this unless she told the police investigators about it. Even then, the analyst would have no way of knowing whether the information was correct and true.

As a result of these uncertainties it was common, when ABO blood typing results represented the best evidence available, for forensic biologists to state conclusions about ABO blood type results conditionally. In this case the scientist might say that the man is excluded prima facie (on the face of it) by the blood group results, but that some conditions would have to be met before the exclusion could be considered absolute.

Saturday, April 9, 2011

ABO Blood Typing and Inclusion

In the late 1970s a woman named Cathleen Crowell-Webb reported a sexual assault in a suburb of Chicago. At the time the case was “routine”; that is, it did not differ from any of the hundreds of such cases that come into forensic laboratories every year. Crowell was found to be a type B secretor, and the suspect in the assault, Gary Dotson, was also a type B secretor

The physical evidence in the case consisted of a visible drainage stain on the complainant’s underwear. This stain was tested and shown to contain semen, and ABO typing showed that it had B and H group substances. The suspect was thus included as a possible source of the semen. After a trial at which Crowell-Webb testified that he was her attacker, Dotson was convicted of sexual assault and sent to prison. The conviction was based on Crowell’s testimony, but also on the testimony of a forensic scientist about the physical evidence just described.

Six years later Crowell, by then married and called Crowell-Webb, announced at a press conference that she had lied about Dotson’s involvement: She had not been sexually assaulted at all, and the semen in evidence belonged to a consensual partner. This action caused a major furor in the press and in the courts. Eventually DNA typing proved that the semen in evidence was in fact not from Dotson but from another man who was Crowell-Webb’s boyfriend at the time of the alleged assault. Dotson was released from prison. Cathleen Crowell-Webb died in May 2008 at the age of 46.

In the scrutiny the case received after Crowell-Webb recanted her trial testimony, it came out that the forensic biologist who had done the original analysis had misinterpreted his findings. He had said that only a B secretor male could be the source of the semen. That is not true, as the following table shows.








The table shows that a male who is a B secretor could be the source of the semen found in the underwear, as the analyst said. But the source could also be a type O secretor or a nonsecretor of any ABO type. Semen from males of any of the three combinations mixed with the secretions of a B secretor female would yield the same results upon typing the mixture.

The reason this point matters is because the expert usually gives the court a percentage of the population who could be sources. Type B secretors are about 7 percent of the Caucasian population and 15 percent of the African-American population. (It is general practice to state the population percentages for different racial groups, because the blood type frequencies are different among the groups.)

But given that the source could also have been an O secretor or a nonsecretor, the sum of those three groups represents about 66 percent of the Caucasian and 85 percent of the African-American populations. Even understanding that 7 percent or 15 percent of the population is a lot of people, there is quite a difference between telling the jury that the defendant is in a population group of potential semen sources that is 7 or 15 percent versus one that is 66 or 85 percent.

This is an example of an inclusion case, the defendant was included as a potential source of the semen found in the evidence. It demonstrates two things: First, including the person as a possible source of the semen stain does not mean he is the source and, second, accurately presenting the findings to the court is important. Telling the court that only 7 percent of Caucasian or 15 percent of African-American males could have been sources was simply wrong and could well have misled the jury.

Friday, April 8, 2011

Dr. Lattes’s Forensic Blood-Typing Cases

In 1916 Dr. Leone Lattes published two cases that illustrated the forensic value of the then new technique for ABO typing bloodstains. Lattes used a method for determining the ABO type of bloodstains that relied on detection of the specific antibodies. Although published 15 years after Landsteiner first described the ABO blood group system in human beings, this work is the first report of ABO typing of dried blood for forensic purposes.

In the first case a man returned home from a trip to another town with what appeared to be bloodstains on his shirt. His wife saw the stains and accused him of adultery during his trip. Though he vehemently denied these accusations, his wife refused to believe him. The man consulted the legal medical institute for help, and Lattes agreed to test the stains to find out whom they might match. The man thought the stains might very well be his own blood, but they could also have been his wife’s or possibly beef blood from the butcher shop. Lattes determined that the stains were human, eliminating the butcher shop possibility, and that they had the same ABO group as the man (type O) but were different from that of his wife (type A). According to Lattes’s account, the findings helped restore peace to this family.

In the second case a man was a suspect in a homicide. He had bloodstains on his coat, but he claimed they were the result of a nosebleed. The suspect was type O, and the victim was type A (determined at the autopsy). The stains were type O, eliminating the victim as a source, and thus exonerating this particular suspect.

Note that because the results of the stains showed a blood type different from that of the victim, the victim could be absolutely eliminated as a possible source. The fact that they had the same blood type as the suspect does not show that they came from the suspect, however, only that they came from someone with blood type O. The results are consistent with the stains having come from the suspect, but they do not prove it by a long stretch. A little less than half the population is type O.

Tests for the ABO antibodies in bloodstains are called Lattes tests. Later, methods were developed to test for the antigens, and Lattes tests became a backup or confirmatory method to determine the blood type in dried bloodstains.

Thursday, April 7, 2011

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.

Wednesday, April 6, 2011

Forensic Botany

There are at least two different ways plant materials can be useful as forensic evidence. One way—perhaps the most obvious—is as “trace” materials. Trace evidence can be many things, including hairs, fibers, glass, soil, and cosmetics, as well as plant materials that might consist of leaves, stems, flowers, seed pods, tree bark, or pollen. Many plants reproduce by producing pollen or spores, which are released to and carried by the wind. Seeds may also be windborne, and the wind can carry leaves, stems, or other plant structures as well. Plant materials often occur on or fall to the ground and can easily be transferred to a person’s shoes or clothing or to a vehicle.

Most plants have a defined geographical habitat, a limited range of territory in which they live. The presence of traces of a given plant on someone or something can help show that the person or object was within the plant’s habitat area. This type of information could provide valuable investigative leads and circumstantial evidence: For example, a suspect in a crime might be associated with a crime scene in this way; or a suspect’s vehicle could be associated with a crime scene; or a suspect could be associated with a crime victim by finding similar plant materials on both their clothing.

With DNA technology there may also be ways to take the presence of plant materials on a person or vehicle a step further in placing the person or vehicle at a location than is possible just using visual botanical comparisons. For instance, in a real-life case the presence of pods from a paloverde tree in the bed of a pickup circumstantially placed the vehicle at the scene of a homicide in Arizona in 1992 (see sidebar “The Denise Johnson Case”).

A second way plant materials can become forensic evidence is when they are ingested by people as food. Medical examiners can sometimes judge how long someone has been dead by the extent of food digestion in the stomach. Food digests in the stomach at a known rate, and forensic doctors know about this. Sometimes the food is recognizable and can be correlated with a witness statement about the decedent’s (the dead person’s) last meal, helping establish time of death. Medical examiners might be able to recognize various plant materials in stomach contents, like seeds, leafy structures, or the skin from a fruit. If someone was with a decedent a short time before death, and especially if they were eating together, the witness can help the medical examiner confirm the contents of a last meal. This information can help a medical examiner determine when the person died. Plant materials that pass through the digestive system but are not well digested, and thus not very much changed, can be recognized and identified in fecal material by forensic plant experts. Forensic botany experts can recognize a plant from the structures they can see when looking at it through a microscope. Although one does not hear about this very often, fecal matter can be evidence at scenes and on clothing, and once in a while a forensic botanical examination can become important in one of these cases.

Tuesday, April 5, 2011

What Bugs Can Say in Death Cases

Entomology is the study of insects; forensic entomology is the study of insects to help resolve legal questions. One of the most important uses of forensic entomology is to help uncover information about certain human deaths.

There are more species (different kinds) of insects than any other group of plants or animals on Earth. They make up nearly 25 percent of all the known living organisms. With so many species there is great competition among insects for food resources; therefore, different groups (families) have developed distinct feeding strategies for their survival. The different groups of insects have evolved over time by selecting specific kinds of foods, which other insects may not compete for; for instance, insects known as corn pests feed only on corn plants, while the hog louse feeds only on pigs, and so on. This process of specialization has brought about the situation where some insects select as their food resource dead, decomposing vertebrate animal carrion (the decaying soft tissues on the animals). These species of insects are known as the carrion insects, and a great many are flies.

When an animal or a human being dies, these carrion insects are attracted to the body by the chemicals that are released into the air during decomposition. Very sensitive chemical sensors on the carrion species detect these chemicals. The flies go to the body in order to lay eggs, generally in moist bodily orifices, such as the nose, mouth, or eye. After a time the eggs hatch, and fly larvae (maggots) emerge and feed on the decomposing body. When they have had enough to eat, the maggots will crawl from the body to seek a place to form a puparium (cocoon), the container for the next stage of the life cycle, the pupa. Eventually the next generation of adult insects (imago) will emerge from these puparia. All insects have this type of life cycle: egg, larva, pupa, adult.

Depending on geographical location, a variety of species of insects will be attracted to and colonize (lay eggs on) human and animal remains. Entomologists know the life cycles of these insects. When the insect forms are observed on and/or recovered from bodies, forensic entomologists can use them to “back calculate” when the eggs were laid. That time is usually very close to the time of death. The elapsed time between death and the discovery of a body is the postmortem interval (PMI).

Among the variables that can affect the precision of the calculations used to determine the time of death from insect life cycles, the most important is temperature. The speed with which the insects progress through their life cycle is temperature dependent. In addition, some of the stages are inactive during darkness, so it is important to know the number of hours of daylight that the carrion insects in a dead body were exposed to. The National Weather Service has stations throughout the country that collect temperature, daylight hours, rainfall, and other such information, but the information is collected at specific time points (such as every hour), and the weather station might be some distance from the location of the body. In some cases the time of death is very important, and the forensic entomologists may the only ones with the tools to pin it down.

It is also known that if a person has ingested drugs, and the drugs are still present in their body, the maggots feeding on that body will also ingest the drugs. The maggots can then be tested by toxicologists to see what drugs were in the body on which they were feeding. This forensic specialty area has been called entomotoxicology.

Another recent development in forensic entomology is the ability to determine human DNA types from blood-feeding insects. Insect groups such as lice, bedbugs, fleas, and mosquitoes, all of which can take a human blood meal, will have in their bodies the blood of the person they have fed upon. Thus, provided that the insects are recovered, these insects may disclose the identity of the person whose blood they were feeding on. In addition, entomologists can analyze the digestive tract contents of maggots that have fed on a decomposing human for a human DNA profile and thereby aid in the identification of the dead human. If a body was left for a time at the death scene and a maggot infestation resulted, maggots left behind after the body was moved from that scene could be used to identify the victim by DNA analysis of the maggots remaining behind.

Sunday, April 3, 2011

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.

Saturday, April 2, 2011

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.

Friday, April 1, 2011

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.

Wednesday, March 30, 2011

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.