Dj dittman cause of death

After studying this chapter you should be able to: Name the parts of a nucleotide and explain how they are linked together to form DNA

Understand the concept of base pairing as it relates to the double-helix structure of DNA

Contrast DNA strands that code for the production of proteins with strands that contain repeating base sequences

Explain the technology of polymerase chain reaction (PCR) and how it applies to forensic DNA typing

Understand the concept of electrophoresis

Understand the structure of an STR

Describe the difference between nuclear and mitochondrial DNA

Understand the use of DNA computerized databases in criminal investigation

List the necessary procedures for the proper preservation of bloodstained evidence for laboratory DNA analysis

DNA: the indispensable forensic science tool

amelogenin gene amino acids buccal cells chromosome complementary base

pairing deoxyribonucleic acid

(DNA) electrophoresis epithelial cells human genome hybridization low copy number mitochondria multiplexing nucleotide picogram polymer polymerase chain

reaction (PCR) primer proteins replication restriction fragment

length polymor- phisms (RFLPs)

sequencing short tandem repeat

(STR) substrate control tandem repeat touch DNA Y-STRs

KEY TERMS

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The discovery of deoxyribonucleic acid (DNA), the deciphering of its structure, and the decod- ing of its genetic information were turning points in our understanding of the underlying concepts of inheritance. Now, with incredible speed, as molecular biologists unravel the basic structure of genes, we can create new products through genetic engineering and develop diagnostic tools and treatments for genetic disorders.

For a number of years, these developments were of seemingly peripheral interest to forensic scientists. All that changed when, in 1985, what started out as a more or less routine investiga- tion into the structure of a human gene led to the discovery that portions of the DNA structure of certain genes are as unique to each individual as fingerprints. Alec Jeffreys and his colleagues at Leicester University, England, who were responsible for these revelations, named the process for isolating and reading these DNA markers DNA fingerprinting. As researchers uncovered new approaches and variations to the original Jeffreys technique, the terms DNA profiling and DNA typing came to be applied to describe this relatively new technology.

This discovery caught the imagination of the forensic science community because forensic scientists have long desired to link with certainty biological evidence such as blood, semen, hair, or tissue to a single individual. Although conventional testing procedures had gone a long way toward narrowing the source of biological materials, individualization remained an elusive goal. Now DNA typing has allowed forensic scientists to accomplish this goal. The technique is still relatively new, but in the few years since its introduction, DNA typing has become routine in public crime laboratories and has been made available to interested parties through the services of a number of skilled private laboratories. In the United States, courts have overwhelmingly admitted DNA evidence and accepted the reliability of its scientific underpinnings.

What Is DNA? Inside each of 60 trillion cells in the human body are strands of genetic material called chromosomes. Arranged along the chromosomes, like beads on a thread, are nearly 25,000 genes. The gene is the fundamental unit of heredity. It instructs the body cells to make proteins that determine everything from hair color to our susceptibility to diseases. Each gene is actually composed of DNA specifically designed to carry out a single body function.

Interestingly, although DNA was first discovered in 1868, scientists were slow to understand and appreciate its fundamental role in inheritance. Painstakingly, researchers developed evidence that DNA was probably the substance by which genetic instructions are passed from one genera- tion to the next. But the major breakthrough in comprehending how DNA works did not occur until the early 1950s, when two researchers, James Watson and Francis Crick, deduced the struc- ture of DNA. It turns out that DNA is an extraordinary molecule skillfully designed to carry out the task of controlling the genetic traits of all living cells, plant and animal.

Structure of DNA Before examining the implications of Watson and Crick’s discovery, let’s see how DNA is con- structed. DNA is a polymer. As we will learn in Chapter 12, a polymer is a very large molecule made by linking a series of repeating units.

NUCLEOTIDES In the case of DNA, the repeating units are known as nucleotides. A nucleotide is composed of a sugar molecule, a phosphorus-containing group, and a nitrogen-containing molecule called a base. Figure 15–1 shows how nucleotides can be strung together to form a DNA strand. In this figure, S designates the sugar component, which is joined with a phosphate group to form the backbone of the DNA strand. Projecting from the backbone are the bases.

The key to understanding how DNA works is to appreciate the fact that only four types of bases are associated with DNA: adenine, cytosine, guanine, and thymine. To simplify our dis- cussion of DNA, we will designate each of these bases by the first letter of their names. Hence, A will stand for adenine, C will stand for cytosine, G will stand for guanine, and T will represent thymine.

Again, notice in Figure 15–1 how the bases project from the backbone of DNA. Also, al- though this figure shows a DNA strand of four bases, keep in mind that in theory there is no limit to the length of the DNA strand; in fact, a DNA strand can be composed of a long chain with millions of bases. The information just discussed was well known to Watson and Crick by

deoxyribonucleic acid (DNA) The molecules carrying the body’s genetic information; DNA is double stranded in the shape of a double helix.

chromosome A rodlike structure in the cell nucleus, along which the genes are located; it is composed of DNA surrounded by other material, mainly proteins.

polymer A substance composed of a large number of atoms; these atoms are usually arranged in repeating units, or monomers.

nucleotide The unit of DNA consisting of one of four bases—adenine, guanine, cytosine, or thymine—attached to a phosphate–sugar group.

C S

P

T S

P

S

P

A S

P

S

S

S

G

FIGURE 15–1 How nucleotides can be linked to form a DNA strand. S designates the sugar component, which is joined with phosphate groups (P) to form the backbone of DNA. Projecting from the backbone are four bases: A, adenine; G, guanine; T, thymine; and C, cytosine.

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the time they set about detailing the structure of DNA. Their efforts led to the discovery that the DNA molecule is actually composed of two DNA strands coiled into a double helix. This can be thought of as resembling two wires twisted around each other.

As these researchers manipulated scale models of DNA strands, they realized that the only way the bases on each strand could be properly aligned with each other in a double-helix configuration was to place base A opposite T and G opposite C. Watson and Crick had solved the puzzle of the double helix and presented the world with a simple but elegant picture of DNA (see Figure 15–2).

COMPLEMENTARY BASE PAIRING The only arrangement possible in the double-helix configuration was the pairing of bases A to T and G to C, a concept that has become known as complementary base pairing. Although A–T and G–C pairs are always required, there are no restrictions on how the bases are to be sequenced on a DNA strand. Thus, one can observe the sequences T–A–T–T or G–T–A–A or G–T–C–A. When these sequences are joined with their complements in a double-helix configuration, they pair as follows:

T A T T G T A A G T C A | | | | | | | | | | | |

A T A A C A T T C A G T

Any base can follow another on a DNA strand, which means that the possible number of dif- ferent sequence combinations is staggering! Consider that the average human chromosome has DNA containing 100 million base pairs. All of the human chromosomes taken together contain about 3 billion base pairs. From these numbers, we can begin to appreciate the diversity of DNA

P

S G C

P

S

P

S T A

P

S

P S G C

P

S

S T A S

C G

T A

FIGURE 15–2 A representation of a DNA double helix. Notice how bases G and C pair with each other, as do bases A and T. This is the only arrangement in which two DNA strands can align with each other in a double-helix configuration.

complementary base pairing The specific pairing of base A with T and base G with C in double-stranded DNA.

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and hence the diversity of living organisms. DNA is like a book of instructions. The alphabet used to create the book is simple enough: A, T, G, and C. The order in which these letters are ar- ranged defines the role and function of a DNA molecule.

DNA at Work The inheritable traits that are controlled by DNA arise out of its ability to direct the production of complex molecules called proteins. Proteins are actually made by linking a combination of amino acids. Although thousands of proteins exist, they can all be derived from a combination of up to 20 known amino acids. The sequence of amino acids in a protein chain determines the shape and function of the protein. Let’s look at one example: The protein hemoglobin is found in our red blood cells. It carries oxygen to our body cells and removes carbon dioxide from these cells. One of the four amino acid chains of “normal” hemoglobin is shown in Figure 15–3(a). Studies of individuals with sickle-cell anemia show that this inheritable disorder arises from the presence of “abnormal” hemoglobin in their red blood cells. An amino acid chain for “abnormal” hemoglobin is shown in Figure 15–3(b). Note that the sole difference between “normal” and “abnormal” or sickle-cell hemoglobin arises from the substitution of one amino acid for another in the protein chain.

The genetic information that determines the amino acid sequence for every protein manu- factured in the human body is stored in DNA in a genetic code that relies on the sequence of bases along the DNA strand. The alphabet of DNA is simple—A, T, G, and C—but the key to de- ciphering the genetic code is to know that each amino acid is coded by a sequence of three bases. Thus, the amino acid alanine is coded by the combination C–G–T; the amino acid aspartate is coded by the combination C–T–A; and the amino acid phenylalanine is coded by the combina- tion A–A–A. With this code in hand, we can now see how the amino acid sequence in a protein chain is determined by the structure of DNA. Consider the DNA segment

–C–G–T–C–T–A–A–A–A–C–G–T–

The triplet code contained within this segment translates into

[C–G–T] – [C–T–A] – [A–A–T] – [C–G–T] alanine aspartate phenylalanine alanine or the protein chain

alanine aspartate phenylalanine alanine

Interestingly, this code is not restricted to humans. Almost all living cells studied to date use the same genetic code as the language of protein synthesis.1

If we look at the difference between “normal” and sickle-cell hemoglobin (see Figure 15–3), we see that the latter is formed by substituting one amino acid (valine) for another (gluta- mate). Within the DNA segment that codes for the production of normal hemoglobin, the letter sequence is

–[C–C–T]–[G–A–G]–[G–A–G]– proline glutamate glutamate

Individuals with sickle-cell disease carry the sequence

–[C–C–T]–[G–T–G]–[G–A–G]– proline valine glutamate

Thus, we see that a single base or letter change (T has been substituted for A in valine) is the un- derlying cause of sickle-cell anemia, demonstrating the delicate chemical balance between health and disease in the human body.

As scientists unravel the base sequences of DNA, they obtain a greater appreciation for the roles that proteins play in the chemistry of life. Already the genes responsible for hemophilia,

WEBEXTRA 15.1 What Is DNA?

proteins Polymers of amino acids that play basic roles in the structures and functions of living things.

amino acids The building blocks of proteins; there are twenty common amino acids; amino acids are linked to form a protein; the types of amino acids and the order in which they’re linked determine the character of each protein.

valine valine

Normal hemoglobin

Sickle-cell hemoglobin

histidine histidine

leucine leucine

threonine threonine

proline proline

glutamate valine

glutamate

1

2

3

4

5

6

7 glutamate

(a) (b)

FIGURE 15–3 (a) A string of amino acids composes one of the pro- tein chains of hemoglobin. (b) Substitution of just one amino acid for another in the protein chain results in sickle-cell hemoglobin.

1 Instructions for assembling proteins are actually carried from DNA to another region of the cell by ribonucleic acid (RNA). RNA is directly involved in the assembly of the protein using the genetic code it received from DNA.

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Duchenne muscular dystrophy, and Huntington’s disease have been located. Once scientists have isolated a disease-causing gene, they can determine the protein that the gene has directed the cell to manufacture. By studying these proteins—or the absence of them—scientists will be able to devise a treatment for genetic disorders.

A 13-year project to determine the order of bases on all 23 pairs of human chromosomes (also called the human genome) is now complete. Knowing where on a specific chromosome DNA codes for the production of a particular protein is useful for diagnosing and treating genetic diseases. This information is crucial for understanding the underlying causes of cancer. Also, comparing the human genome with that of other organisms will help us understand the role and implications of evolution.

Replication of DNA Once the double-helix structure of DNA was discovered, how DNA duplicated itself before cell division became apparent. The concept of base pairing in DNA suggests the analogy of positive and negative photographic film. Each strand of DNA in the double helix has the same informa- tion; one can make a positive print from a negative or a negative from a positive.

The Process of Replication The synthesis of new DNA from existing DNA begins with the unwinding of the DNA strands in the double helix. Each strand is then exposed to a collection of free nucleotides. Letter by letter, the double helix is re-created as the nucleotides are assembled in the proper order, as dictated by the principle of base pairing (A with T and G with C). The result is the emergence of two identical cop- ies of DNA where before there was only one (see Figure 15–4). A cell can now pass on its genetic identity when it divides.

Many enzymes and proteins are involved in unwinding the DNA strands, keeping the two DNA strands apart, and assembling the new DNA strands. For example, DNA polymerases are enzymes that assemble a new DNA strand in the proper base sequence determined by the origi- nal, or parent, DNA strand. DNA polymerases also “proofread” the growing DNA double helices for mismatched base pairs, which are replaced with correct bases.

Until recently, the phenomenon of DNA replication appeared to be of only aca- demic interest to forensic scientists interested in DNA for identification. However, this changed when researchers perfected the technology of using DNA polymerases to copy a DNA strand located outside a living cell. This laboratory technique is known as polymerase chain reaction (PCR). Put simply, PCR is a technique designed to copy or multiply DNA strands in a laboratory test tube.

In PCR, small quantities of DNA or broken pieces of DNA found in crime- scene evidence can be copied with the aid of a DNA polymerase. The copying process is highly temperature dependent and can be accomplished in an auto- mated fashion using a DNA thermal cycler (see Figure 15–5). Each cycle of the PCR technique results in a doubling of the DNA, as shown in Figure 15–4. Within a few hours, 30 cycles can multiply DNA a billionfold. Once DNA copies are in hand, they can be analyzed by any of the methods of modern molecular biology. The abil- ity to multiply small bits of DNA opens new and exciting avenues for forensic scientists to explore. It means that sample size is no longer a limitation in characterizing DNA recovered from crime-scene evidence.

DNA Typing with Short Tandem Repeats Tandem Repeats Geneticists have discovered that portions of the DNA molecule contain sequences of letters that are repeated numerous times. In fact, more than 30 percent of the human genome is composed of repeating segments of DNA. These repeating sequences, or tandem repeats, seem to act as filler or spacers between the coding regions of DNA. Although these repeating segments do not seem

human genome The total DNA content found within the nucleus of a human cell; it is composed of approximately three billion base pairs of genetic information.

replication The synthesis of new DNA from existing DNA.

polymerase chain reaction (PCR) A technique for replicating or copying a portion of a DNA strand outside a living cell; this technique leads to millions of copies of the DNA strand.

Parent DNA unravels

New double helices formed

FIGURE 15–4 Replication of DNA. The strands of the original DNA molecule are separated, and two new strands are assembled.

tandem repeat A region of a chromosome that contains multiple copies of a core DNA sequence that are arranged in a repeating fashion.

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–G–T–C–T–C–A–G–C–T–T–C–C–A–G–

–C–A–G–A

C–C–A–G

–C–A–G–A–G–T–C–G–A–A–G–G–T–C–

The third step is to add the DNA polymerase and a mixture of free nucleotides (A, C, G, T) to the sepa- rated strands. When the test tube is heated to 72°C, the polymerase enzyme directs the rebuilding of a double-stranded DNA molecule, extending the prim- ers by adding the appropriate bases, one at a time, resulting in the production of two complete pairs of double-stranded DNA segments:

–G–T–C–T–C–A–G–C–T–T–C–C–A–G–

C–A–G–A–G–T–C–G–A–A–G–G–T–C–

G–T–C–T–C–A–G–C–T–T–C–C–A–G

–C–A–G–A–G–T–C–G–A–A–G–G–T–C–

This completes the first cycle of the PCR technique, which results in a doubling of the number of DNA mol- ecules from one to two. The cycle of heating, cooling, and strand rebuilding is then repeated, resulting in a fur- ther doubling of the DNA molecules. On completion of the second cycle, four double-stranded DNA molecules have been created from the original double-stranded DNA sample. Typically, 28 to 32 cycles are carried out to yield more than one billion copies of the original DNA molecule. Each cycle takes less than two minutes.

Polymerase Chain Reaction

The most important feature of PCR is the knowledge that an enzyme called DNA polymerase can be di- rected to synthesize a specific region of DNA. In a relatively straightforward manner, PCR can be used to repeatedly duplicate or amplify a strand of DNA millions of times. As an example, let’s consider a seg- ment of DNA that we want to duplicate by PCR:

–G–T–C–T–C–A–G–C–T–T–C–C–A–G–

–C–A–G–A–G–T–C–G–A–A–G–G–T–C–

To perform PCR on this DNA segment, short se- quences of DNA on each side of the region of interest must be identified. In the example shown here, the short sequences are designated by boldface letters in the DNA segment. These short DNA segments must be available in a pure form known as a primer if the PCR technique is going to work.

The first step in PCR is to heat the DNA strands to about 94°C. At this temperature, the double-stranded DNA molecules separate completely:

–G–T–C–T–C–A–G–C–T–T–C–C–A–G–

–C–A–G–A–G–T–C–G–A–A–G–G–T–C–

The second step is to add the primers to the sep- arated strands and allow the primers to combine, or hybridize, with the strands by lowering the test-tube temperature to about 60°C.

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FIGURE 15–5 The DNA Thermal Cycler, an instrument that automates the rapid and precise temperature changes required to copy a DNA strand. Within a matter of hours, DNA can be multiplied a millionfold.

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to affect our outward appearance or control any other basic genetic function, they are nevertheless part of our genetic makeup, inherited from our parents in the manner illustrated by the Punnett square (page 366). The origin and significance of these tandem repeats is a mystery, but to forensic scientists they offer a means of distinguishing one individual from another through DNA typing.

Forensic scientists first began applying DNA technology to human identity in 1985. From the beginning, attention has focused on the tandem repeats of the genome. These repeats can be visualized as a string of connected boxes with each box having the same core sequence of DNA bases (see Figure 15–6). All humans have the same type of repeats, but there is tremendous varia- tion in the number of repeats that each of us has.

Up until the mid-1990s, the forensic community aimed its efforts at characterizing repeat segments known as restriction fragment length polymorphisms (RFLPs). A number of dif- ferent RFLPs were selected by the forensic science community for performing DNA typing. Typically a core sequence is 15 to 35 bases long and repeats itself up to one thousand times. These repeats are cut out of the DNA double helix by a restriction enzyme that acts like a pair of scissors. Once the DNA molecules have been cut up by the restriction enzyme, the resulting fragments were sorted out by separating the fragments by a technique known as electrophoresis.

RFLP DNA typing has the distinction of being the first scientifically accepted protocol in the United States used for the forensic characterization of DNA. However, its utility has been short lived. New technology incorporating PCR has supplanted RFLP. In its short history, perhaps RFLP’s most startling impact related to the impeachment trial of President Bill Clinton. The whole complexion of the investigation regarding the relationship of the president with a White House intern, Monica Lewinsky, changed when it was revealed that Ms. Lewinsky possessed a dress that she claimed was stained with the president’s semen. The FBI Laboratory was asked to compare the DNA extracted from the dress stain with that of the president. An RFLP match was obtained between the president’s DNA and the stain. The combined frequency of occurrence for the seven DNA types found was nearly one in eight trillion, an undeniable link. The dress and a copy of the FBI DNA report are shown in Figure 15–7.

Why couldn’t the PCR technology be applied to RFLP DNA typing? Simply put, the RFLP strands are too long, often containing thousands of bases. PCR is best used with DNA strands that are no longer than a couple of hundred bases. The obvious solution to this problem is to charac- terize DNA strands that are much shorter than RFLPs. Another advantage in moving to shorter DNA strands is that they would be expected to be more stable and less subject to degradation brought about by adverse environmental conditions. The long RFLP strands tend to break apart under adverse conditions not uncommon at crime scenes.

Short Tandem Repeats (STRs) Currently, short tandem repeat (STR) analysis has emerged as the most successful and widely used DNA-profiling procedure. STRs are locations (loci) on the chromosome that contain short sequence elements that repeat themselves within the DNA molecule. They serve as helpful mark- ers for identification because they are found in great abundance throughout the human genome.

STRs normally consist of repeating sequences of three to seven bases; the entire strand of an STR is also very short, less than 450 bases long. These strands are significantly shorter than those encountered in other DNA typing procedures. This means that STRs are much less susceptible to degradation and are often recovered from bodies or stains that have been subject to extreme decomposition. Also, because of their shortness, STRs are an ideal candidate for multiplication by PCR, thus overcoming the limited-sample-size problem often associated with crime-scene

WEBEXTRA 15.2 Polymerase Chain Reaction

prim er A short strand of DNA used to target a region of DNA for replication by PCR.

hybridization The process of joining two comple- mentary strands of DNA to form a double-stranded molecule.

G–C–T G–G–T G–C–T G–G–C C–T–C Fifteen–base core

FIGURE 15–6 A DNA segment consisting of a series of repeating DNA units. In this illustration, the 15-base core can repeat itself hundreds of times. The entire DNA segment is typically hundreds to thousands of bases long.

restriction fragment length polymorphisms (RFLPs) Different fragment lengths of base pairs that result from cutting a DNA molecule with restriction enzymes.

electrophoresis A technique for separating mol- ecules through their migration on a support medium while under the influence of an electrical potential.

WEBEXTRA 15.3 An Animated Demonstration of Gel Electrophoresis

short tandem repeat (STR) A region of a DNA molecule that contains short segments consisting of three to seven repeating base pairs.

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FIGURE 15–7 The dress and the FBI Report of Examination for a semen stain located on the dress.

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Power source Mixtures of DNA fragments of different sizes placed on gel-coated plate

Gel-coated plate

(a)

Power source

Electric potential applied to plate

Substances with an electrical charge migrate across plate

(b)

Power source

Completed gel

Longer fragments move more slowly

Shorter fragments move more quickly

Separated bands allow analyst to characterize DNA in dried blood

(c)

can be separated and identified by electrophoresis. The technique is particularly useful for separating and identifying complex biochemical mixtures. In forensic science, electrophoresis is most useful for character- izing proteins and DNA in dried blood.

Forensic serologists have developed several electro- phoretic procedures for characterizing DNA in dried blood. Mixtures of DNA fragments can be separated by gel electrophoresis by taking advantage of the fact that the rate of movement of DNA across a gel-coated plate depends on the molecule’s size. Smaller DNA fragments move faster along the plate than larger DNA fragments. After completing the electrophoresis run, the separated DNA is stained with a suitable de- veloping agent for visual observation (see Figure 2).

Electrophoresis

Electrophoresis is somewhat related to thin-layer chromatography (discussed in Chapter 9) in that it separates materials according to their migration rates on a stationary solid phase. However, electrophoresis does not use a moving liquid phase to move the ma- terial; instead, an electrical potential is placed across the stationary medium.

The nature of the medium can vary; most forensic applications call for a starch or agar gel coated onto a glass plate. Under these conditions, only substances that possess an electrical charge migrate across the stationary phase (see Figure 1 [a–c]). Because many substances in blood carry an electrical charge, they

inside the science

FIGURE 1

The technique of gel electrophoresis. (a) Applying samples to the plate. (b) Applying electric potential to the plate to cause the fragments to migrate. (c) Separation of the fragments on the gel allows for analysis.

FIGURE 2

DNA fragments separated by gel electrophoresis are visualized under a UV light.

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evidence. Only the equivalent of 18 DNA-containing cells is needed to obtain a DNA profile. For instance, STR profiles have been used to identify the origin of saliva residue on envelopes, stamps, soda cans, and cigarette butts.

To understand the utility of STRs in forensic science, let’s look at one commonly used STR known as TH01. This DNA segment contains the repeating sequence A–A–T–G. Seven TH01 variants have been identified in the human genome. These variants contain 5 to 11 repeats of A–A–T–G. Figure 15–8 illustrates two such TH01 variants, one containing 6 repeats and the other containing 8 repeats of A–A–T–G.

During a forensic examination, TH01 is extracted from biological materials and amplified by PCR as described earlier. The ability to copy an STR means that extremely small amounts of the molecule can be detected and analyzed. Once the STRs have been copied or amplified, they are separated by electrophoresis. Here, the STRs are forced to move across a gel-coated plate under the influence of an electrical potential. Smaller DNA fragments move along the plate faster than do larger DNA fragments. By examining the distance the STR has migrated on the electro- phoretic plate, one can determine the number of A–A–T–G repeats in the STR. Every person has two STR types for TH01, one inherited from each parent. Thus, for example, one may find in a semen stain TH01 with six repeats and eight repeats. This combination of TH01 is found in ap- proximately 3.5 percent of the population. It is important to understand that all humans have the same type of repeats, but there is tremendous variation in the number of repeats each of us has.

When examining an STR DNA pattern, one merely needs to look for a match between band sets. For example, in Figure 15–9 DNA extracted from a crime-scene stain matches the DNA recovered from one of three suspects. When comparing only one STR, a limited number of people in a population would have the same STR fragment pattern as the suspect. However, by using additional STRs, a high degree of discrimination or complete individualization can be achieved.

Multiplexing What makes STRs so attractive to forensic scientists is that hundreds of types of STRs are found in human genes. The more STRs one can characterize, the smaller the percentage of the popula- tion from which these STRs can emanate. This gives rise to the concept of multiplexing. Using PCR technology, one can simultaneously extract and amplify a combination of different STRs.

A A

T G

–

A A

T G

–

A A T G – A A T G –

A A T G – A A T G –

A A

T G

–

A A

T G

– A

A

T G – A A T G – A A T G – A A T G

A A T G – A A T G –

FIGURE 15–8 Variants of the short tandem repeat TH01. The upper DNA strand contains six repeats of the sequence A-A-T-G; the lower DNA strand contains eight repeats of the sequence A-A-T-G. This DNA type is designated as TH01 6,8.

multiplexing A technique that simultaneously detects more than one DNA marker in a single analysis.

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One STR system on the commercial market is the STR Blue Kit. This kit provides the necessary materials for amplifying and detecting three STRs (a process called triplexing): D3S1358, vWA, and FGA. The design of the system ensures that the size of the STRs does not overlap, thereby allowing each marker to be viewed clearly on an electrophoretic gel, as shown in Figure 15–10. In the United States, the forensic science community has standardized 13 STRs for entry into a national database known as the Combined DNA Index System (CODIS).

When an STR is selected for analysis, not only must the identity and number of core repeats be defined, but the sequence of bases flanking the repeats must also be known. This knowledge allows commercial manufacturers of STR typing kits to prepare the correct primers to delineate the STR segment to be amplified by PCR. Also, a mix of different primers aimed at different STRs will be used to simultaneously amplify a multitude of STRs (i.e., to multiplex). In fact, one STR kit on the commercial market can simultaneously make copies of 15 different STRs (see Figure 15–11).

DNA Typing with STRs The 13 CODIS STRs are listed in Table 15–1 along with their probabilities of identity. The probability of identity is a measure of the likelihood that two indi- viduals selected at random will have an identical STR type. The smaller the value of this probability, the more discriminating the STR. A high degree of discrimina- tion and even individualization can be attained by analyzing a combination of STRs (multiplexing). Because STRs occur independently of each other, the probability of biological evidence having a particular combination of STR types is determined by the product of their frequency of occurrence in a population. This combination is referred to as the product rule (see page 64). Hence, the greater the number of STRs characterized, the smaller the frequency of occurrence of the analyzed sample in the general population.

The combination of the first 3 STRs shown in Table 15–1 typically produces a frequency of occurrence of about 1 in 5,000. A combination of the first 6 STRs typically yields a frequency of occurrence in the range of one in two million for the Caucasian population, and if the top 9 STRs are determined in combination, this fre- quency declines to about one in one billion. The combination of all 13 STRs shown in Table 15–1 typically produces frequencies of occurrence that measure in the

FIGURE 15–9 A DNA profile pattern of a suspect and its match to crime-scene DNA. From left to right, lane 1 is a DNA standard marker; lane 2 is the crime- scene DNA; and lanes 3 to 5 are control samples from suspects 1, 2, and 3, respectively. Crime- scene DNA matches suspect #2.

Si ze

M ar

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Control stain

Questioned stain

FGA

vWA

D3S1358

FIGURE 15–10 A triplex system containing three loci: FGA, vWA, and D3S1358, indicating a match between the questioned and the standard/reference stains.

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FIGURE 15–11 STR profile for 15 loci.

TABLE 15–1 The 13 CODIS STRs and Their Probability of Identities

STR African American U.S. Caucasian

D3S1358 0.094 0.075 vWA 0.063 0.062 FGA 0.033 0.036 TH01 0.109 0.081 TPOX 0.090 0.195 CSF1PO 0.081 0.112 D5S818 0.112 0.158 D13S317 0.136 0.085 D7S820 0.080 0.065 D8S1179 0.082 0.067 D21S11 0.034 0.039 D18S51 0.029 0.028 D16S539 0.070 0.089

Source: The Future of Forensic DNA Testing: Predictions of the Research and Development Working Group. Washington, D.C.: National Institute of Justice, Department of Justice, 2000, p. 41.

range of 1 in 575 trillion for Caucasian Americans and 1 in 900 trillion for African Americans. Importantly, several commercially available kits allow forensic scientists to profile STRs in the kinds of combinations cited here.

Sex Identification Using STRs Manufacturers of commercial STR kits typically used by crime laboratories provide one ad- ditional piece of useful information along with STR types: the sex of the DNA contributor. The focus of attention here is the amelogenin gene located on both the X and Y chromosomes. This

WEBEXTRA 15.4 Understand the Operational Principles of Capillary Electrophoresis

amelogenin gene A genetic locus useful for determining gender.

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Criminalistics: An Introduction to Forensic Science, Eleventh Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2015 by Pearson Education, Inc.

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DNA: THE INDISPENSABLE FORENSIC SCIENCE TOOL 389

high-voltage energy. The column is coated with a gel polymer, and the DNA-containing sample solution is injected into one end of the column by applying a high voltage to an electrode immersed in the DNA solution. The STR fragments then move through the column under the influence of an electrical potential at a speed that is related to the length of the STR fragments. The other end of the column is connected to a detector that tracks the separated STRs as they emerge from the column. As the DNA peaks pass through the detector, they are recorded on a dis- play known as an electropherogram, as shown in the figure.

Capillary Electrophoresis

The separation of STRs can typically be carried out on a flat gel-coated electrophoretic plate, as described earlier. However, the need to reduce analysis time and to automate sampling and data collection has led to the emergence of capillary electrophoresis as the pre- ferred technology for characterization of STRs. Capil- lary electrophoresis is carried out in a thin glass column rather than on the surface of a coated-glass plate.

As illustrated in the figure, each end of the col- umn is immersed in a reservoir of buffer liquid that also holds electrodes (coated with platinum) to supply

inside the science

gene, which is actually the gene for tooth pulp, has an interesting characteristic in that it is shorter by six bases in the X chromosome than in the Y chromosome. Hence, when the amelogenin gene is amplified by PCR and separated by electrophoresis, males, who have an X and a Y chromo- some, show two bands; females, who have two X chromosomes, have just one band. Typically, these results are obtained in conjunction with STR types.

Another tool in the arsenal of the DNA analyst is the ability to type STRs located on the Y chromosome. The Y chromosome is male specific and is always paired with the X chromo- some. Although more than 400 Y-STRs have been identified, only a small number of them are being used for forensic applications. One commercial kit allows for the characterization of 17 Y chromosome STRs. When can it be advantageous to seek out Y-STR types? Generally, Y-STRs are useful for analyzing blood, saliva, or a vaginal swab that is a mix originating from more than one male. For example, Y-STRs prove useful when multiple males are involved in a sexual assault. Further simplifying the analysis is that any DNA in the mixture that originates from a female will not show.

Keep in mind that STR types derived from the Y chromosome originate only from this single male chromosome. A female subject, or one with an XX chromosome pattern, does not contribute any DNA information. Also, unlike a conventional STR type that is derived from two chromosomes and typically shows two bands or peaks, a Y-STR has only one band or peak for each STR type.

For example, the traditional STR DNA pattern may prove to be overly complex in the case of a vaginal swab containing the semen of two males. Each STR type would be expected to show four bands, two from each male. Also complicating the appearance of the DNA profile may be the presence of DNA from skin cells emanating from the walls of the vagina. In this circum- stance, homing in on the Y chromosome greatly simplifies the appearance and interpretation of the DNA profile. Thus, when presented with a DNA mixture of two males and one female, Y-STR analysis would show only two bands (one band for each male) for each Y-STR type.

When gauging the significance of a Y-STR match between questioned and known speci- mens, one should take into consideration that all male paternal relatives (e.g., brothers, father, male offspring, and uncles) would be expected to have the same Y-STR profile.

Another advantage of employing STR technology is to extend the success of detecting eviden- tial DNA from vaginal swabs collected from rape victims. Casework experience has demonstrated significant difficulties in obtaining traditional STR DNA profiles for the male donor from vaginal swabs collected after three to four days after intercourse. However, the application of Y-STR tech- nology often extends the routine postcoital detection time to five days for the male donor.

Y-STRs Short tandem repeats located on the human Y chromosome.

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Voltage supplyElectrical potential is applied to STR fragments in column

Capillary column

Injection Area

Detector

Sample containing DNA is injected into capillary column

Platinum-coated electrodes

(a)

Fragments move at different speeds through column under influence of electric potential

Voltage supply

Injection Area

Detector tracks separated STRs as they emerge from column

Electropherogram recorder shows separation pattern of STRs

Detector

Capillary column

(b)

Capillary electrophoresis technology has evolved from the traditional flat gel electrophoresis approach. The separation of DNA segments is carried out on the interior wall of a glass capillary tube that is kept at a constant voltage. The size of the DNA fragments determines the speed at which they move through the column. This figure illustrates the separation of three sets of STRs (triplexing).

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DNA: THE INDISPENSABLE FORENSIC SCIENCE TOOL 391

characterizing badly fragmented strands of DNA. These smaller amplicons are called “miniSTRs.” One manufacturer of STR kits has produced a miniSTR kit designed to amplify eight miniSTRs, seven of which are totally compatible with the CODIS database. The miniSTRs range in size from 71 to 250 bases. A DNA analyst suspecting a degraded sample now has the option, if sample size permits, of running both tradi- tional STR and miniSTR determinations, or just the latter.

The advent of miniSTRs means that forensic sci- entists can now analyze samples that were once thought to be of no value. One of the first benefac- tors of miniSTR technology was the identification of a number of victims from the Waco Branch Davidian fire. Also, a number of World Trade Center victims were identified by miniSTR technology. Another fo- cus of attention has been human hair. In the past, ex- tracting nuclear DNA out of the hair shaft has been enormously difficult; the number of STRs in hair has been found to be very low as well as highly degraded. However, one study has demonstrated that miniSTRs may overcome some of the difficulties in obtaining partial profiles from the degraded DNA present in shed hair.2