Introduction

Biological evidence retrieved from a victim or crime scene can be examined at its most fundamental level — the deoxyribonucleic acid (DNA) molecule. DNA profiling can be used to

1. Establish the link between evidential DNA and that of the possible suspect's

DNA

2. Identify whether the DNA in question is human or nonhuman and establish sex

The purpose of this chapter is to acquaint the reader with the application of DNA and genetic identification techniques in criminal investigation. The information within this section also includes personal interviews and materials furnished by Dr. Robert Shaler1 and Dr. Pasquale Buffolino, Ph.D.2

Several private corporations perform forensic DNA analysis in addition to the FBI laboratory. The private corporations include:

• Forensic Science Associates in California

• Orchid Biosciences with laboratories in Maryland, Tennessee, and Texas

• Roche Biomedical Labs in North Carolina

• ACTG, Inc., in Colorado

I served as an investigative consultant for the Lifecodes Corporation under the supervision of Dr. Robert Shaler. At that time, he was the company's forensic director. Dr. Shaler is the director of the Department of Forensic Biology for the New York Medical Examiner's Office and is a nationally recognized authority on

* Dr. Pasquale Buffolino, who worked in the New York City Medical Examiner's Office under the supervision of Dr. Robert Shaler from 1992 to 2001, prepared and updated this chapter at the request of the book's author to ensure the accuracy of the contents presented herein.

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forensic DNA applications. He led the groundbreaking DNA effort to identify human remains recovered from the World Trade Center attacks.

Dr. Buffolino is the director of the Nassau County Medical Examiner's Office in East Meadow, New York. Dr. Buffolino worked in the New York City Medical Examiner's Office under the supervision of Dr. Shaler from 1992 to 2001; he updated and prepared this DNA chapter at my request to assure the accuracy of its contents.

Deoxyribonucleic Acid — DNA

DNA, or deoxyribonucleic acid, is housed in every nucleated cell in the body. These DNA molecules are often described as the body's blueprints because they carry the genetic codes that govern the structure and function of every component of the body. DNA has been described as the fundamental natural material that determines the genetic characteristics of all life forms. Although some portions of our DNA are relatively conserved through the evolutionary process, as humans we share a human form that is basically human specific, and other classes of organisms share a DNA composition unique to that particular species, e.g., an elephant, horse, cow, insect, fish or mouse.

In fact, the DNA molecule carries the genetic information that establishes each person as separate and distinct. (The exceptions are identical twins.) We as humans create progeny through the transfer of this DNA to our children. According to the genetic experts, the DNA molecule's configuration does not vary from cell to cell. Therefore, the billions of cells that make up each person contain the same molecules of DNA carrying the same codes in precisely the same sequence. All cells in the human body are nucleated except for red blood cells.

The Cell

The cell is the basic unit of all living organisms, including humans, animals, insects, and plants. The human body has more than 10 trillion cells. The cell is composed of two parts:

1. The nucleus, which contains two structures: the chromosomes and the nucleoli

2. The cytoplasm, which is all of the material inside the cell membrane outside of the nucleus

Molecular Biology of the Cell

Before forensic applications of DNA technology can be discussed, one must have a general understanding of the cell. A cell is the basic unit of life for all living creatures. Whether they are simple single cell organisms or complex organisms containing billions of cells, as humans do, they function to give life. If one would

Figure 16.1 COMMONLY ENCOUNTERED CELL TYPES IN CRIMINAL INVESTIGA-

TIONS. (A) Graphic representation of a magnified blood sample under light microscopy. White blood cells or lymphocytes (represented in blue stain) contain a single nucleus per cell. There are four types: neutrophils, basophils, eosinophils, and monocytes. Each type performs a different function in the immune response. (B) Graphic representation of stratified squamous epithelium cells (under light microscopy) commonly found on the surfaces subject to abrasion as skin, inner cheek (buccal cells), vagina, and anus. The cells are constantly sloughing off and regenerating. This example was prepared from a buccal cell swabbing. (C) Graphic representation of a human sperm cell. Sperm is equipped with a tail known as the flagellum, which propels it through aqueous media, enabling it to deliver DNA to the egg. The flagella are driven by a motor situated in the midpiece, which is rich in mitochondrial DNA. Mitochondrial DNA supplies the motor the energy it requires to function. Sperm cell DNA is packaged in the nucleus situated in the head region. (Information by Dr. Pasquale Buffolino, Ph.D., director of Department of Forensic Genetics, Nassau County Medical Examiner's Office. Figure courtesy of Medical Legal Art. Illustration copyright 2005, Medical Legal Art, www.doereport.com.) take a handful of beach sand and sift through it until one grain of sand remained, this grain would represent the most basic unit of that beach from which the sand was collected, similar to a single human cell.

Cells work together to form units, which differentiate into tissue and organs. Human cells (also known as eukaryotic cells) are highly compartmentalized structures composed of three major units: a cell membrane, cytoplasm, and a nucleus. Human cells are bound by a structure known as the cell membrane, which protects it from its environment. Within this membrane resides a viscous substance known as the cytoplasm. Composed mainly of water, the cytoplasm contains all membranebound structures, called organelles, two of which are the nucleus and the mitochondrion. The nucleus, also a membrane-bound structure, is where our nuclear DNA is located and functions as the information center of the cell. DNA, which is responsible for our genes and the factors that control them, is packaged within the nucleus in the form of chromosomes.

Not all cells contain a nucleus. A general misconception is that DNA is carried in red blood cells. Red blood cells, which function to transport oxygen to our body tissue, are non-nucleated cells. DNA is carried in the nuclei of a variety of cell types; for the purposes of this discussion, we will limit them to (1) white blood cells, or lymphocytes, responsible for the production of antibodies that protect us from infection; (2) epithelial cells, which line the inner and outer surfaces of our body; and (3) sperm cells involved in reproduction. Biological fluids containing nucleated cells most commonly encountered in connection with violent crimes are blood, semen, and saliva. This is not to say that no other types of fluids or biological matter may be present (e.g., pus, perspiration, urine, vomit, fecal matter, skin cells from points of contact).

Structure and Function of DNA

Nuclear DNA

DNA is commonly referred to as the hereditary material of life due to its role in transmitting genetic information from generation to generation. Nuclear DNA is inherited in a diploid fashion, with half of our DNA inherited from our mothers and the remaining half from our fathers. DNA molecules are packaged into tightly wound structures known as chromosomes. If DNA were stretched out, it would measure approximately 5 feet and exceed the capacity of the nucleus; therefore, DNA must associate with proteins known as histones, which package the DNA into chromosomes. Normal humans contain 46 chromosomes organized as 23 pairs. The 23rd pair is specific to the sex chromosomes, where an X or Y chromosome is inherited from the father and an X chromosome is inherited from the mother. In its native form, DNA exists as a double-stranded helix composed of a series of nitrogenous bases known as nucleotides and a sugar and phosphate backbone. There are four bases: two purines — adenine (A) and guanine (G) — and two pyrimadines — thymine (T) and cytosine (C).

If one could envision the DNA helix uncoiled, it would resemble a ladder with the sides composed of the sugar and phosphate strands and the rungs created from the bases. The native form of DNA is kept in place by base pair complementation, which is an association through hydrogen bonding. The bases do not pair randomly. Adenine always binds with thymine and guanine always binds with cytosine to form A–T, T–A, G–C, and C–G base pairs. The human genome contains approximately 6 billion bases, the sequences of which determine the genetic make-up of our cells. The entire genome was sequenced in 2003 as part of the Human Genome Project, a 13-year study which has led to major medical and forensic advancements.

Figure 16.2 CHEMICAL COMPOSITION OF DNA. Chemical structure of purine bases adenine (A) and guanine (G). Chemical structure of pyrimadine bases cytosine (C) and thymine (T). Chemical structure of deoxyribonucleotide. The deoxyribonucleotide binds to one of the four bases at the N–H* position to form a deoxyribonucleic acid unit. (Courtesy of Medical Legal Art. Illustration copyright 2005, Medical Legal Art, www.doereport.com.)

Figure 16.3 DNA MOLECULE. (Left) Double-stranded DNA in its uncoiled form resembling a ladder structure. Representation of A–T G–C base pairing. (Right) DNA in its native helical form. (Courtesy of Medical Legal Art. Illustration copyright 2005, Medical Legal Art, www.

doereport.com.)

Approximately 99.9% of our entire genome has been conserved through the evolutionary process; therefore, normal humans share the same genetic sequence within these conserved regions. In order to survive, humans must consume, break down, and convert foods into energy. This process is crucial to our existence and is therefore conserved genetically. Mutations or variations within genes are known as polymorphisms (defined as "many forms").

Mutations within vital genes result in the alteration of the particular protein for which the gene codes and thus alter its intended function, affecting the organism's ability to survive. Therefore, evolution plays a significant roll in molecular polymorphisms. Because conserved regions within the human genome have no discriminative value, they would not serve as probative forensic markers.

The remaining 0.1% of our DNA (approximately 6 million bases) displays molecular polymorphisms or variations from person to person. These polymorphisms result in multiallelic variants. An allele is defined as an alternative form of a gene. Therefore, a multiallelic variant defines many forms of the same gene. For example, the hair color gene contains several forms: black, brown, blond, red, etc. The same gene codes for hair color; however, there are several forms of the particular gene. There are two types of polymorphisms: (1) sequence polymorphisms and (2) length polymorphisms. Sequence polymorphisms display variations within the genetic sequence composition. Length polymorphisms display variations within the physical length of DNA.

Figure 16.4 DNA POLYMORPHISM. (A) Sequence polymorphism. The maternal (M) and paternal (P) alleles are represented. Base position number (8) denoted below the cursor displays a polymorphic site or single nucleotide polymorphism (SNP). The specific sequence at this given region of DNA (also known as a "DNA locus") can vary from individual to individual. Therefore, individuals can be distinguished by their genetic variation in sequence. (B) Length polymorphism. Display of a short tandem repeat region (STR) where a short sequence of DNA (in this example, AATG) is repeated randomly, four repeats in the maternal allele and six repeats in the paternal allele. (C) Graphic representation of the length polymorphism described in (B). Each block () represents a tetrameric AATG repeat. Length polymorphisms differ in the number of times a particular sequence block is repeated at a locus. Therefore, individuals can be distinguished by their respective length of DNA. (Information by Dr. Pasquale Buffolino, Ph.D., director of Department of Forensic Genetics, Nassau County Medical Examiner's Office. Figure courtesy of Medical Legal Art. Illustration copyright 2005, Medical Legal Art, www.doereport.com.)

Approximately 5% of the human genome codes for genes. The remaining 95% has no genetic function and is often referred to as "nonsense" or "junk" DNA because its function is not entirely understood. These polymorphic DNA regions are known to repeat over and over like a stutter. Contrary to their role in gene production, these noncoding regions have been found to contain a substantial degree of length polymorphism. These regions are termed variable number of tandem repeats (VNTRs) for their multiallelic variation in randomly repeated sequences of DNA; genetic researchers have discovered that they are unique to the individual. The first hypervariable region of DNA was discovered by chance. Following the discovery of several other such regions, Dr. Alec Jeffreys, an English geneticist, developed a method that could detect many hypervariable regions simultaneously.3 This method was essentially the first individual-specific identification system — what is commonly referred to today as "DNA fingerprinting."

Further research determined that the human genome is full of repetitive DNA. These multiallelic markers are categorized according to the number of tandem repeat units within them. There are three classifications: (1) satellite DNA, which contains repeat blocks of greater than 100 base pairs; (2) minisatellite DNA, which contains repeat blocks of 10 to 100 base pairs; and (3) microsatellite DNA, which contains repeat blocks of 2 to 9 base pairs commonly known as short tandem repeats (STRs).4,5

Y-Chromosomal DNA

The human Y chromosome is approximately 60 million base pairs in size. Unlike nuclear DNA, Y chromosomal DNA is passed from father to son and is transferred along the paternal lineage, commonly referred to as a haploid fashion of inheritance. The Y chromosome plays a central role in human biology. The presence or absence of this chromosome determines gender. Therefore, the presence of a Y chromosome in a developing embryo results in a male child, and those without it become female.6 Approximately 50% of its genome comprises repetitive DNA. More than 200 microsatellites and a single minisatellite are identified on the Y chromosome. The degree of variation within these markers is comparable to their autosomal (non-sex chromosomes) counterpart and therefore has made the genetic analysis of the Y chromosome a valuable tool in forensic identity testing.4

Mitochondrial DNA

In addition to nuclear DNA, eukaryotes contain another form of DNA, which resides within the mitochondrion. Human mitochondrial DNA (mtDNA) is a circular genome composed of 16,569 base pairs and codes for 37 genes, mainly responsible for energy production within the cell. This makes the mitochondrial genome approximately 4000 times smaller than the Y chromosome. mtDNA is inherited in a haploid fashion, strictly from our mothers, making mtDNA the Y chromosome female counterpart. Therefore, all individuals along the same maternal lineage share a common mtDNA type. The complete nucleotide sequence of the extranuclear genome was reported in 1981.7

Base-substitution mutation rates of mtDNA have been calculated at approximately ten times the rate of nuclear DNA resulting in a high degree of genetic diversity. The displacement loop (D-loop), which is situated within the mitochondrial control (noncoding) region, is one of the most polymorphic regions in the entire genome. Two regions of increased polymorphism were discovered through sequence comparison of the 680-bp (base pairs) D-loop region among human, bovine, and rat genomes. These regions are referred to as the hypervariable region I and hypervariable region II. There are several hundred to thousands of mitochondria per cell, with multiple copies of the mtDNA genome per mitochondrion as compared with one nucleus and one copy of the nuclear genome per cell. Therefore, in highly degraded samples, the chance of obtaining mtDNA is greater than it is for nuclear DNA. These inherent characteristics (maternal inheritance, high degree of molecular polymorphism, and high copy numbers) make this genome a powerful alternative to nuclear DNA testing.8

Techniques and Procedures in Forensic DNA Analysis

Collection and Preservation of Biological Evidence

The most crucial role in the examination of biological evidence is its method of collection and preservation. The integrity of forensic evidence has always been the burden of the crime scene investigator, who must assure that proper evidence collection procedures are followed because improper collection and preservation may compromise DNA evidence. Because there is a chance that biological evidence may contain hazardous pathogens such as the hepatitis B virus and the human immunodeficiency virus (HIV), crime scene investigators must be trained in the methods of universal precautions. What is noteworthy is that the same precautions that protect the crime scene investigator from biological pathogens also protect the integrity of DNA evidence.

At the least, the use of disposable Tyvex jump suits, respirators, and latex examination gloves will minimize the chances of introducing DNA contaminants. DNA contamination can be defined as the introduction of nonrelated DNA from an external source into a DNA sample relevant to the crime. The latest DNA technology, known as the polymerase chain reaction (PCR), utilizes trace amounts of DNA; therefore, the potential of contamination must be addressed by crime scene investigators and laboratory personnel. Sterile techniques should always be employed even if the crime scene investigator has been summoned to a "nonDNA" scene. There is no guarantee that physical evidence initially collected for the sole purpose of criminalistics (e.g., latent fingerprints) will not require DNA analysis at a later date.

There are essentially three methods of collection: (1) swabbing, (2) cutting, and (3) recovery of the entire item. Each method has a specific purpose and should be utilized in an effort to preserve forensic evidence. Any instruments used in the collection of evidence (i.e., scissors, razors, tweezers, etc.) must be sterilized prior

Figure 16.5 EVIDENCE TECHNICIAN AT CRIME SCENE WEARING TYVEX SUIT. This photos depicts a properly outfitted ERT technician processing a bloodstain in the crime scene. (Courtesy of Detective Mark Czworniak, Chicago Police Department.)

Figure 16.6 EVIDENCE TECHNICIAN SWABBING BLOODSTAIN. Close-up photo of an

ERT swabbing a bloodstain in a crime scene. (Courtesy of Detective Mark Czworniak, Chicago Police Department.)

to the collection of each sample unless disposable devices are used. The use of a 10% bleach solution followed by a 90 to 100% ethanol solution is the most common method of sterilization. Because of its versatility and reliability, the most common method of collecting DNA evidence is swabbing. This method can be used for liquid and dry bloodstains from nonporous surfaces. Sterile, individually wrapped cotton-tipped applicators are commercially available for this method. For dried stains, the cotton swab should be moistened with sterile water. When a sufficient sample exists, the entire cotton swab (or multiple swabs) should be used to collect the sample. For small stains or control samples of nonstained areas less than 1/8 in., the sample should be concentrated at the tip of the cotton swab. These samples may not be visible, especially if the sample collected does not cause the swab to stain.

As a general rule, forensic biologists sample the top third of swabs submitted to the laboratory; therefore, concentrating the sample on the tip of the swab assures it will be maximized for DNA analysis. Biological evidence should be collected by cutting when swabbing or collection of the entire item is not possible, especially when dealing with porous surfaces (i.e., fabric) that may prevent sufficient samples from being collected by the swabbing method. Stains from clothing recovered from victims, suspects, or witnesses should not be removed because the stain may be part of a pattern that could be more probative than the DNA evidence. For these instances, the collection of the entire item is recommended.

Figure 16.7 BLOODSPATTER PATTERN. The right sleeve of a bloodstained shirt worn by a defendant during the commission of a stabbing is represented. The victim was fatally stabbed through the left lung and heart. When questioned by case detectives, the suspect claimed to have been nowhere near the vicinity of the victim during the stabbing and that another gang member was responsible. Submission of the entire item enabled the association of the victim's DNA profile with a distinct bloodspatter pattern. The bloodspatter pattern circled was theorized to have been produced by expiratory blood from the victim. Because this type of bloodspatter cannot be projected for long distances, the suspect's statement was refuted. When confronted with the evidence, the suspect admitted to his role in the crime and accepted the plea bargain offered to him. This case exemplifies collection and submission of the entire item for DNA analysis. (Information from Dr. Pasquale Buffolino, Ph.D., director of Department of Forensic Genetics, Nassau County Medical Examiner's Office.)

Also, at some scenes blood and other relevant biological fluids are not detected on probative items. The crime scene investigator should consider collecting the entire item for a more thorough examination under the controlled environment of the laboratory.

Regardless of the method of collection, all stains, swabs, and all other wet evidence must be thoroughly air dried before packaging. Crime scene investigators are aware that this is sometimes not an easy task, especially for blood-soaked items. Packaging samples wet in airtight containers (e.g., plastic containers) promotes bacterial degradation of DNA and thus the potential for sample loss. Wet swabs should be air dried completely before packaging or packaged in commercially available kits that promote drying.

Large, blood-soaked items may be packaged in a plastic container to protect others from hazardous material, which can soak through paper packaging, but only for the purposes of transport. The item must be air dried completely, preferably under a biological fume hood to advance the drying process, and then packaged properly. To prevent cross-contamination between items, each item must be packaged separately and sealed in a manner to prevent loss or deleterious change from occurring.

Scheduling Analysis

Advancements in forensic DNA technology have led to a significant increase in the number of samples collected during crime scene investigations. For this reason, investigators and attorneys should discuss the probative nature of evidence submitted to the laboratory. There should be a general understanding of how each item relates to the crime and analysis should be prioritized according to the level of importance. Considerations should be made to minimize the testing of nonprobative case evidence because this type of evidence creates extreme bottlenecks in DNA laboratories and its forensic relevance seldom outweighs the cost of analysis.

In most cases, items recovered in connection with violent crimes will require a battery of forensic examinations. Considerations must be made concerning the preservation of all types of evidence — latent, trace, ballistics, DNA, etc. — because one type of examination may destroy another. For example, a firearm should not be submitted to ballistics prior to DNA analysis if there is sufficient cause to believe that blood can be recovered from within the barrel of the weapon. Firing the weapon would greatly reduce the chances of recovering blood, whereas swabbing for the presence of blood will have no effect on ballistics.

One of the most important relationships with the forensic DNA laboratory is that with the latent fingerprint laboratory. The two forensic disciplines must function symbiotically for the benefit of latent and DNA evidence because DNA profiles can be developed from fingerprints. Points of entry (doors, windows, etc.) are common areas dusted for fingerprint evidence. DNA from these areas would be difficult to recover without identification of prints through latent examination. Several studies disprove the detrimental effect of dactyloscopic methods on the ability to develop DNA profiles.9,10

Therefore, in most cases, latent print examination should be performed before DNA analysis because DNA swabbing will destroy latent prints. In any situation where one form of examination may consume another form of forensic evidence, investigators, attorneys, and laboratory personnel must determine which science will result in the greater probability of success and at the same time offer sufficient probative value. Open lines of communication are extremely important in the preservation of forensic evidence, and therefore, submitting agencies should make every effort to inform the laboratory of the nature of their evidence and have a general understanding of the analysis they request.

DNA Extraction

DNA from biological samples recovered from crime scenes must be liberated from the cell prior to DNA analysis using a specific method known as DNA extraction. Essentially, DNA must be separated from other cellular material in a series of steps aimed to purify the DNA molecule. Three general types of DNA extraction methods are commonly used in DNA analysis: (1) organic extraction, (2) Chelex extraction, and (3) magnetic bead extraction. Each method varies mechanistically, and the type chosen is dependant upon the type of sample being examined.

One of the oldest and most common DNA extraction methods is the organic extraction method. In this method, the cell walls and nuclear membranes are disrupted through the use of enzymes, which break down proteins involved in maintaining their structural integrity, and a detergent that is equally disruptive to the membrane. The DNA is then isolated by adding a mixture of organic solvents, which forces the DNA into the top aqueous layer. This layer is transferred to a filtration cartridge where the DNA is purified and concentrated. The method is long and laborious but results in a high yield of undegraded (high molecular weight) DNA and is useful when dealing with low-level, degraded DNA samples.

Geberth11 presents a graphic representation of the original DNA process for clarification and understanding of the DNA extraction process prior to the current STR/PCR technology (see Figure 16.9). This results in an electropherogram instead of an audioradiograph, illustrating how DNA technology has evolved in the new millennium.

The Chelex extraction method is a simpler, more rapid method compared with the organic extraction method. Introduced in 1991,12 the method requires fewer steps, thus minimizing the chances of cross-contamination. A crude yet stable product of low molecular weight DNA is obtained. Chelex extraction does have certain drawbacks because the extracted product is not as pure or concentrated as organically extracted DNA. The method is quick and reliable, especially for simple samples such as bloodstains or buccal swabs submitted as known comparison samples for victims and suspects.

One of the most efficient extraction procedures available to forensic DNA laboratories is the magnetic bead extraction. This technology has been available since the early 1990s; however, it was not a major application in forensic DNA laboratories until the introduction of robotic workstations. In this method, a mag-

Figure 16.8 EXTRACTION. A forensic DNA analyst extracts DNA from a bloodstain of unknown source from an item of evidence. (Courtesy of Fred Drummond, chief of Forensic Sciences, Westchester County, New York, Forensic Laboratory. Photographer: Carlos Morales.) netic bead is coated with a highly specific DNA-binding surface that has no affinity for other cellular contents or PCR inhibitors commonly associated with the substrates of crime scene evidence. Following a short incubation period, the magnetic bead–DNA complex is placed into a magnet, where it is immobilized to the side of the reaction tube. The supernatant (liquid) containing all but DNA is removed from the reaction, leaving behind a highly purified DNA extract. The major advantage of this type of extraction is that it is easily automated and can be used for all types of forensic specimens.

Figure 16.9 ORIGINAL EXTRACTION PROCESS. The DNA–Print™ process. (Courtesy of Lifecodes Corporation, Stamford, Connecticut.)

Figure 16.10 THE QIAGEN M48 BIOROBOT. The Qiagen M48 BioRobot is designed to make the extraction of DNA samples less time consuming. The robot automatically transfers reagents and samples from tube to tube using magnetic beads to hold the DNA and allow its purification. (Courtesy of Fred Drummond, chief of Forensic Sciences, Westchester County,

New York, Forensic Laboratory. Photographer: Keith Mancini.)

Differential DNA Extraction

Forensic evidence recovered in connection with sexual offenses is collected with the use of standardized rape kits. The swabs utilized to recover evidence most often contain a mixture of DNA from the victim (epithelial cells from the vaginal wall) and assailant (sperm cells).13 These cell mixtures can be separated using a differential analysis procedure commonly employed in forensic casework.14 With this procedure, DNA from the semen donor can be separated from the victim's DNA using modifications of the extraction methods discussed previously. The method takes advantage of the robustness of the sperm cell membrane and enables the independent typing of the semen donor's DNA.

DNA Quantification

Following the extraction procedure, the amount of isolated DNA must be quantified. Determining the concentration of DNA in a sample extract is essential for the PCR process. Validation experiments have shown that specific concentrations of DNA are required per reaction to achieve optimal results. Three types of quantification methods employed in modern forensic laboratories are (1) slot blot hybridization, (2) chemiluminescent microtiter plate assay, and (3) quantitative PCR (qPCR).

The most common method currently used in forensic laboratories is the slot blot method. A sample of extracted DNA is immobilized on a nylon membrane using a slot blot apparatus equipped with a vacuum source. The DNA extract is loaded into a well of the apparatus and forced through the membrane by suction. DNA standards of known quantity are included in the test with the sample extracts. In a process known as DNA hybridization, a human- and primate-specific DNA probe is introduced to the immobilized DNA and binds it. The amount of DNA is determined through a reaction which causes the DNA to turn into a visible blue band. An alternative to this colorimetric reaction is a process known as chemiluminesce, where chemicals are used that cause DNA to give off a wavelength of energy, which exposes x-ray film similar to the common medical x-ray. The DNA is quantified by comparing the unknown samples with the known DNA standards.

The second method, chemiluminescent microtiter plate assay, can be performed in a variety of manners; however, this discussion will be limited to the Promega AluQuant™ Human DNA Quantitation System. This system has three advantages over the conventional slot blot hybridization method: (1) human specificity, (2) sensitivity, and (3) amenability to robotic automation. AluQuant functions through a series of reactions that produces a precursor (adenosine triphosphate or ATP) that powers a light-emitting reaction. The amount of light emitted is relative to the amount of DNA in the sample and, as with the slot blot hybridization method, the samples are compared to known DNA standards. DNA concentrations are calculated by an instrument known as a luminometer, which measures the amount of light emitted by the samples.

The most sensitive and accurate method of DNA quantification is qPCR. Using real-time PCR (RT-PCR) technology pioneered by Applied Biosystems (ABI), trace

Figure 16.11 QUANTIBLOT™. The Quantiblot is a procedure to approximate the quantity of DNA present in a sample. Standards are loaded into the top row of the Quantiblot and forensic samples are placed in the rest of the wells. The amount of DNA present in a sample is proportional to the intensity of the color band produced in each sample well relative to the standards. (Courtesy of Fred Drummond, chief of Forensic Sciences, Westchester County, New York, Forensic Laboratory. Photographer: Carlos Morales.)

quantities of DNA can be quantified. Because the system uses sequence-specific probes labeled with fluorescent tags, several regions of DNA can be quantified simultaneously or multiplexed. In one reaction, total genomic DNA, Y-chromosomal DNA, and mtDNA can be quantified because each region of DNA is tagged with a different fluorescent color detected by the RT-PCR instrument. This system has had tremendous impact on the forensic community because it is able to detect male-specific DNA at an increased level of sensitivity as compared with the identification of sperm or semen using the methods of conventional serology.

DNA Amplification (Polymerase Chain Reaction)

PCR was discovered in 1983 by an American chemist named Kerry Mullis, who was awarded a Nobel Prize in 1993. The theory of PCR was derived from the cell's ability to replicate DNA. With this technology, unlimited copies of DNA can be

Figure 16.12 REAL TIME PCR. "Real time PCR" is a new technique used to determine the quantity of DNA present in a sample relative to a set of standards. It works by amplifying the DNA in each sample with fluorescent primers. The change of fluorescence is recorded by camera and analyzed by a computer. The calculated value is used to determine the amount of amplifiable DNA present in a sample. (Courtesy of Fred Drummond, chief of Forensic Sciences, Westchester County, New York, Forensic Laboratory. Photographer: Keith Mancini.)

Figure 16.13 THERMAL CYCLER. The thermal cycler is used to carry out the polymerase chain reaction (PCR) for replication of forensic DNA samples. (Courtesy of Fred Drummond, chief of Forensic Sciences, Westchester County, New York, Forensic Laboratory. Photographer: Keith Mancini.)

Figure 16.14 GRAPHIC REPRESENTATION OF THE PCR PROCESS. One cycle of PCR is represented. DNA is first separated during the denaturation step (step 1) by heating the reaction to 94 to 95°. Now in single-stranded form, DNA primers can bind to the regions of interest on each strand during the annealing step (step 2). Once bound, the primers are extended by Taq DNA polymerase (step 3). At this particular phase of extension, DNA polymerase has added six bases to the top strand and seven to the bottom strand and is in the process of adding the next base (A for both strands). The reaction will continue, repeating the three steps of each cycle, for approximately 30 cycles, producing 100 billion copies of the specific region of DNA. (Information by Dr. Pasquale Buffolino, Ph.D., director of Department of Forensic Genetics, Nassau County, New York, Medical Examiner's Office. Figure courtesy of Medical Legal Art. Illustration copyright 2005, Medical Legal Art, www.doereport.com.)

created from trace quantities of DNA in the laboratory using an instrument known as a thermal cycler. This breakthrough in technology has revolutionized forensic science and the way in which crimes are investigated and solved.

PCR is a three-step process involving repetitive cycles of heating and cooling of the DNA sample reaction according to a preprogrammed set of temperatures. In a single tube, all of the components required to replicate 100 billion copies of a DNA template are added. The reaction requires isolated DNA; fluorescenttagged sequence-specific primers, which bind and flank to the DNA of interest; all four nucleotide bases in the form of deoxynucleotide triphosphates (dNTPs); a solution containing salts, which stabilize the reaction; and an enzyme known as Taq DNA polymerase, which drives the reaction. Amplification occurs by the following process:

Step 1: Denaturation. Prior to the duplication process, double-stranded DNA must be separated into single strands by a process known as denaturation.

Figure 16.15 SCHEMATIC REPRESENTATION OF THE DNA DUPLICATION PROCESS

OF PCR. Following four cycles of PCR, eight identical copies of the original DNA region are produced. Because DNA is replicated in exponential fashion, cycles five, six, and seven will produce 16, 32, and 64 copies, respectively. At the completion of a 30-cycle reaction, greater than 1 billion copies are duplicated. (Information by Dr. Pasquale Buffolino, Ph.D., director of Department of Forensic Genetics, Nassau County, New York, Medical Examiner's Office. Figure courtesy of Medical Legal Art. Illustration copyright 2005, Medical Legal Art, www.doereport.com.)

Denaturation occurs by heating the DNA sample to a temperature between 94 and 95°C for approximately 45 seconds or longer.

Step 2: Annealing of primers to template DNA. Small segments of single-stranded DNA approximately 20 bases in length, known as DNA primers, are constructed to bind specific regions of DNA by cooling the sample to a precalculated temperature. The temperature is crucial to this step because lower than optimal temperatures will result in sequence artifacts and temperatures too high will result in no amplification products.

Step 3: Extension of DNA primers. Extension is carried out at an optimal temperature (72 to 78°C) for the duplication of DNA. The reaction is driven by a heat-stable enzyme known as Taq polymerase, which places the correct base into the elongating strand at a rate of approximately 2000 bases per second. Following approximately 30 cycles of denaturation, annealing, and extension, sufficient copies of template DNA are available for the detection of amplification product and the development of DNA profiles.

Multiplex PCR Analysis

Developing a genetic profile at a single region of DNA would not be sufficient to link individuals to crimes because the probability that multiple individuals share the same genetic profile at a single region is high. Therefore, several regions must be analyzed. To facilitate this, a system known as multiplex PCR analysis is used in forensic casework. Discovered in 1998, multiplex PCR enables the simultaneous amplification of multiple regions of DNA in a single PCR process.15

STR Multiplex PCR Analysis

Multiplex PCR analysis of STR DNA is the most commonly used technology in forensic DNA laboratories today. The relatively small size of STR markers, along with the capability of combining several DNA loci into a PCR multiplex, has made them well suited for the analysis of highly degraded DNA samples associated with forensic casework. Several multiplex PCR systems are commercially available to analyze autosomal and Y-chromosomal DNA. The Applied Biosystems (ABI) AmpFLSTR® Cofiler™, AmpFLSTR® Profiler Plus™, and AmpFLSTR® Identifiler™ systems offer the simultaneous amplification of 7, 10, and 15 autosomal STR loci, respectively. These autosomal systems have been designed to support the 13 CODIS specific DNA loci included in the Federal DNA Database (FGA, vWA, CSF1PO, TH01, TPOX, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, and D21S11). The Promega Corporation PowerPlex® Y System offers the simultaneous amplification of 12 Y-chromosomal STR loci.

Amplification of mtDNA

Although the process of mtDNA analysis differs from STR DNA analysis, the hypervariable regions of mtDNA must also be amplified from trace quantities of DNA extracted from crime scene samples. Because mtDNA differs in base sequence composition and not in length as STR DNA does, the precise genetic base composition must be determined using a process known as cycle sequencing. Cycle sequencing is performed in the same thermal cycler used for PCR.

DNA Detection and Analysis

Detection of amplified fragments can be done in a variety of manners; however, the most popular format is capillary electrophoresis (CE). CE is a method capable of separating amplified fragments of DNA by size; smaller fragments migrate faster than larger fragments through a narrow capillary that functions as a "molecular sieve." An electrical current is applied across the capillary, which causes the negatively charged DNA fragments to migrate toward the anode or positive electrical field. As the fluorescent-labeled fragments migrate through a laser field, they are excited, giving off a specific emission recorded by a camera and sent to a computer workstation used to analyze the data. The CE method for DNA separation is fairly new and relatively simple.

More importantly, the entire procedure is completely automated, beginning from sample injection to result output. Depending upon case-throughput requirements, CE instrumentation is available in single-sample injection or multiplesample injection formats. The majority of forensic laboratories utilize CE instrumentation manufactured by ABI (ABI Prism Genetic Analyzer series).

STR DNA Analysis

DNA fragment data recorded on the ABI Prism Genetic Analyzer CE series is analyzed using genetic software supplied by ABI. Genetic data are recorded in the

Figure 16.16 SCHEMATIC REPRESENTATION OF CAPILLARY ELECTROPHERESIS. Capillary electrophoresis is used to separate DNA fragments based upon their size. An electrical current is applied through a thin tube known as a capillary that acts as a molecular sieve. Negatively charged DNA molecules migrate through the capillary towards the anode (+) with smaller DNA fragments migrating faster than larger fragments. As the fragments move through the detector, their tags are excited by a laser, which causes them to fluoresce. The fluorescent emission is sent to a computer workstation where data are acquired and analyzed. Electronic data are recorded in a form known as an electropherogram. (Information by Dr. Pasquale Buffolino, Ph.D., director of Department of Forensic Genetics, Nassau County, New York, Medical Examiner's Office. Figure courtesy of Medical Legal Art. Illustration copyright 2005, Medical Legal Art, www.doereport.com.)

form of peaks, which represent the STR repeat length for each fragment. The repeats are determined electronically by comparing the migration of the fragments to internal standards.

The autosomal multiplex systems discussed have the inherent capability of gender typing along with the ability to determine an individual, specific DNA profile at 13 forensically informative regions supported by CODIS. The PowerPlex

Y System has the ability to determine a male specific DNA profile at 12 STR regions.

mtDNA Analysis

mtDNA analysis can also be performed on the ABI Prism Genetic Analyzer; however, the detection of sequence data differs from that for STR data. In this system, each

Figure 16.17 CAPILLILARY ELECTROPHERSIS UNIT. Forensic analyst Jennifer Reilly places samples in the ABI Pism 310 capillary electropheresis (CE) unit. This instrument will analyze fragments of DNA to obtain a genetic profile. (Courtesy of Fred Drummond, chief of Forensic Sciences, Westchester County, New York, Forensic Laboratory. Photographer: Keith Mancini.)

base of mtDNA is recorded as a fluorescent-labeled peak, with each peak representative of the specific base in the fragment of mtDNA. The complete base composition of the mitochondrial region is compared with a mitochondrial reference sequence known as the Cambridge reference sequence,16 and any differences between this reference and the evidentiary material are recorded with known samples.

STR DNA Databases

Population Statistics

The statistical significance of a DNA match must be considered because there are two possibilities for a match: (1) the DNA profile from the crime scene sample originated from the suspect; or (2) the DNA profile originated from someone other than the suspect who consequently has the same DNA profile as the suspect. If the profile observed is uncommon within the population, the probability of the first scenario is more likely than the second. If the profile is common within the population, the second scenario is possible, depending upon its frequency of occurrence.

Profile frequencies are calculated using population databases constructed from a random group of individuals categorized according to their ethnicity (e.g., Caucasian, Black, Hispanic, Asian). Profile frequencies for autosomal STRs are expressed in terms of random match probabilities or the chance that some individual other than the included (i.e., the suspect) would possess the same DNA profile. Similar databases exist for Y-chromosomal STRs and mitochondrial DNA; however, frequencies are not calculated the same as for autosomal STR databases. Results are expressed in the number of times a particular haplotype has been observed in the database (e.g., three observances in 100,000 samples searched). Therefore, estimates of haplotype rarity depend upon the size of the haplotype database used.

Figure 16.18 ELECTROPHEROGRAM. DNA analysis can be used to compare body fluid stains. In this sample, a bloodstain was found on a suspect's shirt. The evidence sample, samples from the victim and suspect, is submitted for DNA analysis. The resultant electropherogram shows that the DNA profile from the blood on the suspect's shirt matches the victim's DNA profile. (Courtesy of Fred Drummond, chief of Forensic Sciences, Westchester County, New York, Forensic Laboratory.)

With the STR systems employed today, population frequencies far greater than the population of the Earth (approximately 6.2 billion people) are calculated. The magnitude of these estimates is such that it is unlikely that two people, excluding identical twins, would possess the same DNA profile. As a result, laboratories have been reporting, with reasonable scientific certainty, that an included individual is definitively the source of a biological sample.17 This method is commonly referred to as "source attribution."

CODIS

The national DNA database known as the Combined DNA Index System (CODIS) began as a 14-state pilot study in 1990. The program expanded nationally as a result of the 1994 DNA Identification Act (Public Law 103 322) giving the FBI legal authority to establish a DNA database for the nation's criminal justice system. Today, all 50 states participate in the CODIS program, which is composed of two main DNA indices: a forensic index that contains DNA profiles developed from crime scene-related evidence and a convicted offender index containing DNA pro-

Figure 16.19A AmpFLSTR® Profiler Plus™ DNA FRAGMENT ANALYSIS. Graphic representation of Profiler Plus electropherogram data collected on an ABI 310 Prism Genetic Analyzer and analyzed with ABI GenoTyper™ analysis software. The Profiler Plus multiplex system amplifies ten DNA regions (loci) simultaneously. The name of each region of DNA is specified above its peak data. The peaks are representative of the number of repeat units contained in the amplified STR fragment. Because we inherit one allele from our mothers and one from our fathers, a normal individual has two DNA alleles at any given DNA locus. For example, the individual represented in this elecropherogram contains a 14- and 15-repeat fragment in D3S1358 (repeat length represented below each peak ""). If the individual inherits the same allele from his mother and father (i.e., 13 from mother and 13 from father), a single peak is detected (i.e., D8S1179). The gender of the individual can also be determined from the Profiler Plus amplification system. In this example, the amelogenin locus displays an X chromosome consistent with a female individual. (Courtesy of Dr. Pasquale Buffolino, Ph.D., director of Department of Forensic Genetics, Nassau County, New York, Medical Examiner's Office.)

Figure 16.19B AmpFLSTR® COfiler™ DNA FRAGMENT ANALYSIS. Graphic representation of COfiler electropherogram data collected on an ABI 310 Prism Genetic Analyzer and analyzed with ABI GenoTyper™ analysis software. The COfiler multiplex system amplifies seven DNA regions (loci) simultaneously. The theoretical basis for this system is identical to the Profiler Plus amplification system. In this example, the amelogenin locus displays an X and Y chromosome consistent with a male individual. As a quality control measure, two regions within this system (D3S1358 and D7S820) overlap with Profiler Plus (refer to Figure 16.8 top). (Courtesy of Dr. Pasquale Buffolino, Ph.D., director of Department of Forensic Genetics, Nassau County, New York, Medical Examiner's Office.)

Figure 16.19C PowerPlex™ Y System DNA FRAGMENT ANALYSIS. Graphic representation of PowerPlex Y system electropherogram data collected on an ABI 310 Prism Genetic Analyzer and analyzed with the Promega Corporation PowerTyper™ genotype analysis software. The PowerPlex Y System amplifies 12 male-specific Y STR regions (loci) simultaneously. The peak results are recorded in the same manner as in the ProfilerPlus and Cofiler systems (refer to Figure 16.8). (Courtesy of Dr. Pasquale Buffolino, Ph.D., director of Department of Forensic Genetics, Nassau County, New York, Medical Examiner's Office.)

Figure 16.20 MITOCHONDRIAL DNA (mtDNA) SEQUENCE ANALYSIS DATA. (A)

Sequence data corresponding to 40 bases within the region of mtDNA examined. Each peak is representative of a fluorescently labeled base recorded during electrphoresis. (B) Sequence alignment of forensic data (unknown skeletal remains) to the Cambridge Reference Sequence. Results are recorded as differences (polymorphisms) to the reference sequence. (Information by Dr. Pasquale Buffolino, Ph.D., director of Department of Forensic Genetics, Nassau County, New York, Medical Examiner's Office. Figure courtesy of Medical Legal Art. Illustration copyright

2005, Medical Legal Art, www.doereport.com.)

files from qualified convicted offenders. A qualified convicted offender is an individual who has been convicted of a crime in federal, state, and/or local courts where the applicable law permits establishment of a DNA record for the convicted person.

The two indices are searched automatically for matching DNA profiles. The FBI, which funds the program, requires that all participating laboratories utilize the same 13 STR. CODIS is structured as a three-tier hierarchy: a local DNA index system (LDIS), a state DNA index system (SDIS), and a national DNA index system (NDIS). With this approach, each participating laboratory can manage its profiles in accordance with its legal requirements and, at the same time, compare its profiles electronically with other local and state laboratories and the federal laboratory.

National Missing Persons DNA Database

DNA databases can also be used to determine the identity of a missing person. Ideally, the individual's DNA would be the most practical sample to determine an identity; however, DNA from missing persons is not always available. In these types of cases, familial DNA from parents can be used to identify individuals through DNA. A National Missing Persons DNA Database has been established through CODIS. DNA from unidentified remains is compared with a database of DNA samples given voluntarily by family members to establish genetic links.

The Choice of Analysis

The method of analysis is determined based upon the type and quality of the evidentiary material encountered and, ultimately, the ability to develop a DNA profile. Samples can be grouped into three categories: (1) nonmixture/high-level DNA, (2) mixtures/high-level DNA, and (3) highly degraded/low-level DNA. Quite often, an experienced scientist can place a sample into one of these specific categories. This ability is critical to the preservation of biological evidence. When this is not possible, the standard flow of evidence processing will dictate the type of analysis required. An example of a case analysis workflow is described in the flow chart in Figure 16.21.

Admissibility of DNA Evidence

The Frye rule18 is the legal standard of admissibility of evidence for many states. The Frye test requires that scientific testimony meet a "general acceptance" standard and that the procedures used to produce scientific evidence to be introduced at trial have gained general acceptance within the relevant scientific community. Frye was a case in 1923 involving a homicide where a polygraph test was ruled inadmissible. The court in this case held that

Just when a scientific principle or discovery crosses the line between the experimental and demonstrable stages [is] difficult to define. Somewhere in this twilight zone the evidential force of the principle must be recog-

Figure 16.21 CASE ANALYSIS FLOW CHART. The analysis scheduled is ultimately dependent upon the type and quality of evidence encountered. Nonmixture/nondegraded evidence (high-level DNA evidence) is usually analyzed for autosomal STRs. Lower level samples, like samples collected from points of contact, can be considered for Y-STR and mtDNA analysis if they fail to yield a full STR DNA profile. Mixed samples, generally those encountered in sexual assaults, are analyzed for autosomal STRs when sufficient male DNA is available. If the samples encountered contain insufficient amounts of male DNA or if male–male mixtures are involved, Y-STR analysis improves the ability to develop a deducible DNA profile. mtDNA analysis is reserved for nonmixed/highly degraded DNA samples. The inherent capabilities of mtDNA compensate for the degradation and can amplify mtDNA from items such as hair shafts, which are void of nuclear DNA. (Courtesy of Medical Legal Art. Illustration copyright 2005, Medical Legal Art, www.doereport.com.)

nized, and while courts will go a long way in admitting expert testimony deduced from a well-recognized scientific principle or discovery, the thing from which the deduction is made must be sufficiently established to have gained general acceptance in the particular field in which it belongs.

In 1993, Daubert v. Merrell Dow Pharmaceuticals19 rejected the Frye test as the basis for review of novel scientific evidence. Daubert involved a lawsuit against a pharmaceutical company for the antinausea prescription drug Bendectin. The plaintiffs (two children and their parents) alleged that the mother's prenatal ingestion of Bendectin was the cause of her children's birth defects. In Daubert, the court concluded that the plaintiff's experts had not based their testimony on experimental data and that there was insufficient cause to show that ingestion of Bendectin was a risk factor of birth defects. As a result of Daubert, scientific evidence must be supported by validation to establish the reliability of the test and its results. Daubert also granted the trial judge a "gatekeeper" role with respect to the admissibility of scientific evidence. The court in this case held that

The Rules — especially Rule 702 — place appropriate limits on the admissibility of purportedly scientific evidence by assigning to the trial judge the task of ensuring that an expert's testimony both rests on a reliable foundation and is relevant to the task at hand. The reliability standard is established by Rule 702's requirement that an expert's testimony pertain to "scientific...knowledge," since the adjective "scientific" implies a grounding in science's methods and procedures, while the word "knowledge" connotes a body of known facts or of ideas inferred from such facts or accepted as true on good grounds. The Rule's requirement that the testimony "assist the trier of fact to understand the evidence or to determine a fact in issue" goes primarily to relevance by demanding a valid scientific connection to the pertinent inquiry as a precondition to admissibility.

The Daubert ruling is based upon an interpretation of the Federal Rules of Evidence (FRE 702, Testimony by Experts) and focuses on the principles and methodology of the science and not the results they generate.

The choice of legal standard of admissibility, Frye vs. Daubert, depends upon the individual state. The Frye test remains the rule in many states; others have adopted Daubert and others have rejected both and substituted their own rules for admissibility.

Laboratory Accreditation

In 1994, the DNA Identification Act was passed, which resulted in the formation of a DNA Advisory Board (DAB) composed of professionals with expertise in DNA technology, law, and ethics. In 1997, the DAB submitted their recommendations of quality assurance standards for DNA testing laboratories to the director of the FBI. On October 1, 1998, the FBI director issued the first Quality Assurance Standards for DNA Testing Laboratories. The most recent issue, July 1, 2004, includes standards for convicted-offender DNA databasing laboratories.

The Quality Assurance Working Group of the Scientific Working Group on DNA Analysis Methods (QA-SWGDAM) has been organized to maintain and revise the standards set forth.

Quality assurance (QA) is a system of monitoring, auditing, and testing that ensures scientific accuracy. Quality control (QC) is the program which designs the set of measures taken to assure that accurate results are continuously obtained. Laboratory accreditation is an integral part of a DNA laboratory's QA/QC system. The American Society of Crime Laboratory Directors/Laboratory Accreditation Board (ASCLD/LAB) is the laboratory-accrediting body in the U.S. The program is voluntary; however, a few states, such as New York, mandate participation. Participation is an opportunity for laboratories to demonstrate that their management, personnel, operational and technical procedures, equipment, and physical facilities meet established standards. Along with the standards set forth by the ASCLD/LAB governing body, the accreditation process incorporates the FBI Quality Assurance Audit standards. Laboratory accreditation status is essential to DNA testing laboratories and all criminal laboratories because it assures excellence and accuracy.

Forensic DNA Case Studies

Victim Identification

DNA profiling has been successfully used in a number of criminal investigations to positively identify the deceased. In one particular case, Lifecodes' scientists were able to confirm that the brain matter found in a missing woman's vehicle belonged to the victim, who was identified through her DNA and the DNA of her parents. Skeletonized remains (bone marrow), body parts, and other materials that contain nucleated cells can be analyzed for DNA and provide authorities with identification. The following case history is based upon a personal interview with Chief John Dotson, Sparks Police Department, Nevada, formerly a major with the Wichita, Kansas, Police Department.

Case History: Identification of Remains

In December of 1987, Wichita homicide detectives encountered a bizarre crime. The suspect in this case had allegedly killed his wife by putting her into a crematorium. Police were alerted to this possible crime by a civilian complainant, who was suspicious of a bucket left in her garage. The police recovered burnt remains consisting of a number of small fragments of bone and burned flesh, which the medical examiner, Dr. William Eckert, determined to be human. A forensic anthropologist was also brought in to assist the police and medical examiner's office in establishing an identity.

(A) (B)

Figure 16.22 CREMATORIUM. Interior view of the crematorium used to cremate the victim alive. (A) The crematorium operating in a normal fashion. (B) The malfunctioning of the crematorium. Note that only one burner is operating. The killer was unaware of the second burner's malfunction. (Courtesy of retired Major John Dotson, Wichita, Kansas, Police Department and now chief in Sparks, Nevada.)

The homicide investigation revealed that the offender thought that he had planned the perfect murder. His reasoning, according to witnesses, was "No body — no crime." Police learned that he had lured his former wife to a shopping mall on the pretense of shopping together for Christmas presents for their child. At the time, he had been involved in a bitter custody battle over this child with his ex-wife. Somehow, he managed to get his wife to the funeral home, where he was employed as an usher. No one knows whether she was dead or alive, but he somehow managed to place her body into the crematorium and activate the furnace.

Figure 16.23 CREMATORIUM CRIME SCENE PROCESS. Interior view showing the crime measurements along with the dried blood of the victim. (Courtesy of retired Major John Dotson, Wichita, Kansas, Police Department and now chief in Sparks, Nevada.)

Figure 16.24 DRIED BLOOD INSIDE CREMATORIUM. An arrow points to the dried blood of the victim on the inside wall of the crematorium. This blood was collected by authorities and submitted to the Lifecodes Corporation, which provided positive identification of the deceased after comparing and matching the dried blood to the whole blood samples of the victim's parents. (Courtesy of retired Major John Dotson, Wichita, Kansas, Police Department and now chief in Sparks, Nevada.)

The crematorium could reach temperatures of 1500°F and it would take approximately 2 1/2 hours to reduce the body to ashes. However, the suspect did not know that one of the burners was not working properly.

He realized that he was running out of time. He was not even authorized to operate the crematorium. The suspect then decided to get the evidence of the burning out of the crematorium and out of the building before his employer found out. He tried to clean up, but was left with a bucket full of cremains. This bucket of cremains was subsequently seized by Wichita police, which resulted in the suspect's arrest. The cremains provided circumstantial evidence of this brutal slaying. In addition, a unique piece of jewelry, which the deceased wore, was found in the catch basket of the crematorium. However, prosecutors wanted more.

The District Attorney's Office decided on establishing the victim's identity beyond the jewelry and anthropological evidence. A partial femur bone, as well as tissue from the pelvis and blood scrapings from the crematorium was found, along with whole-blood samples of the victim's parents.

Lifecodes Corporation extracted DNA from the blood scrapings from the crematorium and they were able to compare this with the DNA from the whole-blood samples of the victim's parents. Identification of the remains was positively established through the DNA–PRINT identification test, which was in actuality a paternity type of testing.

Case History: Kinship Analysis (Forensic Maternity)

On December 26, 2001, a 25-year-old female admitted herself into a local Nassau County, Long Island, New York, hospital emergency room claiming to have given birth to a stillborn child. She arrived carrying a plastic bag that held a shoebox containing the deceased child wrapped in towels. After a thorough examination revealed a full-term male infant, the Nassau County Police Department Homicide Squad was notified by the hospital, spurring a criminal investigation. During questioning of the suspect, she claimed not to have known of her pregnancy, but admitted to delivering the stillborn child on Christmas day and then placing it into a plastic bag and putting it under her bed. She did not seek medical attention until the following day.

An autopsy conducted by the Nassau County Medical Examiner's Office concluded that the child was born full term and its demise occurred postnatal. The cause of death was determined to be postnatal asphyxia from an unattended delivery at home. This was sufficient evidence to rule the manner of death homicide. These findings led to the arrest and conviction of the defendant in May of 2003. Due to a technical error by the trial court in the manner in which a deliberating juror was replaced, the conviction was vacated by the trial court, and a new trial was ordered. The defendant was released on bail pending her retrial.

In preparation for the retrial, the assistant district attorney (ADA) and lead investigator began reinterviewing witnesses acquainted with the defendant. The defendant was employed at a restaurant as a waitress. A coworker recalled her suspicions that the defendant appeared to be pregnant while working at the restaurant during the spring of 2001. In fact, this coworker, who was employed as the establishment's bartender, refused to serve the defendant alcohol due to her suspicion. The defendant was then reported to have taken a 2-week leave of absence in the beginning April 2001 for an ongoing health problem. What the witness found to be unusual was that when the defendant returned to work, she no longer displayed the physical characteristics associated with pregnancy. Following this meeting, the ADA and investigator stopped at a local diner for lunch.

Figure 16.25 ILLUSTRATION OF SYMBOLIC KINSHIP. Kinship analysis between the two infants was performed using the symbolic kinship software DNA View.20 (Figure courtesy of Medical Legal Art. Illustration copyright 2005, Medical Legal Art, www.doereport.com.)

During this meeting, one of the most damming pieces of evidence against the defendant was uncovered. The ADA recalled being present at a scene April 26, 2001 where a newborn child was discovered packaged in three plastic bags located in the rear parking lot of a restaurant. This restaurant was located within four blocks of the defendant's former residence. The autopsy conducted on this infant concluded that the cause of death was due to intrauterine fetal demise. The connection between the two cases seemed quite far from a relative coincidence, so the ADA immediately contacted the forensic DNA laboratory.

The request for genetic relatedness between the April 2001 and December 2001 incidents was made in December of 2003. The laboratory had in its possession dried bloodstains prepared from postmortem blood recovered during autopsy and muscle tissue from both infants. Bed sheets recovered from the place of residence where the December 2001 birthing took place were also submitted to the laboratory. What was lacking was a known DNA standard from the defendant, which was required for a direct maternity comparison to the April 2001 infant. Due to the nature of the investigation, the ADA chose not to obtain a known DNA standard from the defendant until there was sufficient genetic evidence to make a connection between the two infants and the presumed mother. Based upon the available evidence, the laboratory proceeded in the following manner: (1) the genetic profiles developed from the infants' blood would be compared for sibling relatedness, and if a genetic link was established, (2) the genetic profile developed from the defendant's blood recovered from the bed sheets would be compared with the April 2001 infant's DNA profile to establish maternity.

Kinship analysis between the two infants was performed using the symbolic kinship software DNA View,20 asking the question, "Are the two infants full siblings, half siblings, or are they unrelated?" The sibling test resulted in a likelihood ratio which indicated a common mother and a sibling index, which strongly favored half siblings represented in pedigree B. The genetic link was now established between the two infants. With this probative piece of information, the laboratory proceeded to determine the possibility of a maternal link between the defendant and the April 2001 infant. The maternity index proved maternal relatedness with a 99.9% degree of scientific certainty. Sufficient cause to obtain a DNA standard from the defendant was now established.

In a January 2004 court hearing regarding pretrial issues, the ADA presented the genetic information in his possession. The court ruled that the prosecution would be permitted to introduce evidence of the April 2001 birth to refute the defendant's claim the she did not know she was pregnant when she delivered the December 2001 baby. In June 2004, the defendant admitted guilt by stating that she was aware that the child was alive following birth, but regardless of the circumstance, she placed the child in a plastic bag and put it under her bed without seeking medical attention. The defendant pled guilty to murder in the second degree, waived her right to appeal, and is currently serving an indeterminate sentence of 18 years to life.

Case History: Male-Specific Y-STR Analysis

On June 26, 1998, a once prominent 81-year-old jazz dancer and runway model was found strangled to death in her second-floor Manhattan apartment. The victim, who lived alone with her dog, required the aid of a heath care worker because of her declining health. She was discovered following 3 days of unsuccessful attempts at entering the apartment by her aide. With concern for her patient, she notified the building superintendent, who could not gain access to the apartment because the victim had double locked her door. The New York City Police Department Emergency Services Unit was then notified. The first responders gained access into the apartment using the second-floor fire escape that led them through a living room window. A glass top coffee table located beneath this window was found shattered and the entire apartment's contents had been turned over, giving the impression of an apparent burglary.

The victim was found in the bathroom, kneeling face down in the bathtub and wearing a housecoat, underpants pulled down around her ankles, and a fur coat draped over her body. A dog's leash, still wrapped around the victim's neck, had been used to strangle her. The autopsy performed on the victim determined the manner of death to be the result of ligature strangulation. Notable skin slippage, insect activity, and the accounts of the health care worker placed the time of death approximately 3 days prior to the discovery of the victim.

The crime scene investigation provided an abundance of forensic evidence. Investigators were able to lift fingerprints from several areas of the apartment, including prints from significant items; prints were lifted from a jewelry box that apparently had been disturbed during the burglary. A dried secretion on the back of the victim's thigh was recovered along with the fur coat, which contained a possible dried semen stain that had transferred from the victim to the inner lining. Because a sexual assault was suspected, the medical examiner's office used a sexual assault evidence collection kit during the autopsy.

The initial investigative lead developed from the fingerprint evidence identified a suspect within days of the crime through the state automated fingerprint identification system (SAFIS). Case detectives, unable to locate the suspect, questioned his mother, who informed them that she was unaware of his whereabouts and had not seen him for some time. Days later she was able to persuade her son to turn himself in. During his interrogation, the suspect claimed to be unaware of the incident. He claimed to have been standing out in front of the apartment building where he was confronted by a black male carrying a bag filled with women's clothing and jewelry, looking for help selling the items.

When confronted with the fingerprint evidence placing him at the scene, the suspect changed his account. He then claimed to have been asked by the black male to follow him into the apartment through the fire escape and living room window. While in the apartment, he stated that he observed the victim slumped into the bathtub and noticed a white substance on the back of her thigh he believed was semen. He then claimed that the black male individual became extremely excited over the presence of the victim and began to masturbate. When asked if he was ever intimately involved with the victim, the suspect became extremely offended and swore to have never harmed her. He was sympathetic to her because of her age and health and would occasionally help with her groceries; this gave him an alibi for his presence in the apartment. At the conclusion of the interview, the suspect had made a perplexing comment stating that the detectives would never find any individual who witnessed him committing the crime. With some fortune, the district attorney was able to incarcerate the suspect on July 6, 1998 due to a parole violation, allowing the police the legal right to question him regarding the homicide.

At the laboratory, forensic DNA testing was under way. The presence of semen was confirmed on several items, including the fur coat lining. Autosomal STR testing performed on the semen-positive items yielded two types of results: (1) profiles consistent with the victim and (2) negative results. These results could be explained by an overwhelming concentration of female DNA in the mixture of victim and assailant's DNA21 or an insufficient concentration of sperm cell DNA in nonmixture samples, respectively. The latter has been observed in degraded samples and also in samples contributed by azospermatic assailants.13 Based upon the case circumstances, male-specific Y-STR DNA analysis was employed. The male profile developed from the semen recovered from the lining of the fur coat was compared with the DNA profile developed from the suspect and the black male implicated by the suspect. The relative chance or population frequency of the profile was determined to be 1 in 200 male individuals. Though statistically insignificant, this was sufficient evidence to exclude the black male implicated by the suspect while inculpating the suspect as the semen donor.

With these incriminating data, the suspect was questioned once again and asked to explain the presence of his DNA on the victim. His story changed to that of a long-term consensual relationship with the 81-year-old victim. He claimed that on the day of the incident, he had intercourse with the victim and shortly after she drew a bath for herself. While she was in the bathroom, he became so excited they had intercourse once again and that was when he had ejaculated on the back of her thigh. When he left the apartment, the victim was alive and well. Therefore, the black male he had previously implicated must have entered the apartment following his exit and murdered and burglarized the victim. His accounts may have been convincing to him; however, they did not convince a Manhattan jury, which sentenced him on January 24, 2002, for murder in the first and second degrees along with burglary in the second degree. He is currently serving a life sentence without the possibility of parole.

Case History: Forensic Mitochondrial DNA Analysis

On July 8, 1985, a plastic surgeon reported his 29-year-old wife missing to the New York City Police Department Missing Person Squad, 1 day after she ran out of their apartment following a domestic dispute. The husband claimed that he and his wife had an argument on the morning of July 7th and that she had left furiously for Central Park at around 11:00 A.M. to sunbathe. He had remained home until 5:30 P.M. waiting for her to return and then left for his parents' house. The following week, several messages were left for the husband, but the case detective did not receive a reply until July 14th. On that day, during a second interview, the husband was asked whether anyone had seen his wife leave the apartment the day she disappeared. He claimed that the doorman had told him that he had seen her leave shortly after 11:00 A.M. Up to this point, a full investigation had not uncovered a single piece of physical evidence.

On May 21, 1987, a highly decomposed torso wrapped in a brown plastic bag floated up on the shores of Staten Island. The arms had been chopped off and leg bones removed. The x-rays taken of the torso were compared with a chest x-ray of the missing person developed prior to her disappearance at a Long Island, New York, hospital. An abnormality of the spine and rib cage noted in both x-rays led to a positive identification of the victim. The x-rays were then reviewed by an independent expert, who came to the same conclusion. In September of 1997, the Manhattan District Attorney's Office and New York City Police Department concentrated their efforts in solving the disappearance of this woman, a 12-yearold cold case. The investigation continued with interviews of relatives and close friends of the missing person and her husband. It was determined that there was constant fighting and domestic abuse. The husband would become enraged over menial issues. On one occasion, the husband became so angered when he found his wife smoking a cigarette on the balcony that he strangled her to unconsciousness. The following day, bruise marks resembling fingers were observed on her neck by her employer. This had not been the first time she had been strangled by her husband. Following this incident (and all other prior incidents), the husband begged for her forgiveness and promised that he would never do it again. On November 12, 1983, persuaded by her sister, she filed a complaint with the police department.

The victim's hopes of a sound marriage came to an end around the beginning of 1985 when she began contemplating leaving her husband. Because of the constant abuse, she was frightened that she would be killed during one of his rages. Soon after, the victim received a letter from her psychiatrist warning her that she was in danger from her husband and advising that she separate from him immediately. The victim told her doctor that she intended to use the letter as leverage in obtaining satisfaction during her divorce proceedings. If she did not receive a favorable decision, she was going to tarnish her husband's career with it and threaten to expose allegations of a multimillion dollar insurance fraud involving him and his father. The victim was last seen the afternoon of July 6, when she met with her husband's sister to inform her that she was going to leave him that weekend and was in the process of looking for an apartment.

During the course of the investigation, an overwhelming amount of circumstantial evidence incriminating the husband was uncovered. The most damning piece of evidence was the suspect's conflicting statements regarding the afternoon of July 7, the day his wife was reported missing. On one occasion, the suspect stated that he had waited for his wife in their apartment after she had walked out following their argument and then left for a family birthday party in New Jersey at around 6:00 that same evening. On a second occasion, the suspect claimed to have waited for a few hours and then left for Central Park to find her. During one interview, he claimed to have found nothing, but on a second occasion he claimed to have found a towel and book belonging to the victim.

What the suspect failed to mention was that he was a pilot and on the afternoon of July 7, 1985 he rented a small aircraft from a New Jersey airport between the hours of 4:30 and 7:30 P.M. During this period, the plane's engine was engaged for 1 hour and 56 minutes, giving the suspect sufficient opportunity to fly well over the Atlantic Ocean and dump the victim's body. This conflicted with the suspect's statements; he could not have left for a birthday party at 6:00 P.M. if he was still in flight. With this, the prosecution had sufficient circumstantial evidence to support the crime of homicide.

In 1998, at the time the case was being prepared for grand jury, the forensic laboratory was in the process of validating mitochondrial DNA (mtDNA) testing. Because there had been a confirmed match through x-ray comparisons, a bone sample from the torso was used for a nonprobative case study. Approximately 10 years prior to this, the same sample had been sent out to a private DNA testing laboratory. The results of the nuclear DNA test were negative and therefore no additional testing was performed based upon the degree of sample degradation. Using mtDNA technology, the laboratory was able to develop an mtDNA profile from the bone sample. When the results were compared with the sister of the victim, the laboratory uncovered a major discrepancy: the mtDNA results excluded the torso as a maternal relative of the victim's sister. Therefore, the torso could not be the missing individual and there must have been a misidentification with the x-ray comparisons. Doubts concerning the exclusion were placated when a private testing laboratory confirmed the results. The potential of a sample mix-up during autopsy was then speculated, but also ruled out by exhumation of the torso and retesting of another section of bone. The misidentification was confirmed through mtDNA testing.

Without any physical evidence, particularly the absence of a body, the case went to trial based upon circumstantial evidence in September of 2000. The prosecution presented sufficient evidence to convince the jury that on July 7, 1985, the suspect killed his wife in a violent rage, dismembered her body, and dumped it over the Atlantic Ocean. On October 24, 2000, the suspect was convicted of murder in the second degree and is currently serving an indeterminate sentence of 20 years to life.

Conclusion

DNA and genetic fingerprinting represent the most important breakthrough in crime detection since the discovery of the fingerprint. DNA technology represents the future of forensic medicine and the experts have only begun to scratch the surface with this technology. It is a powerful tool, which protects the innocent just as surely as it pinpoints the guilty. Genetic identification takes the "gamesmanship" out of the trial — either the defendant committed the crime or he did not.

References

1. Dr. Shaler. Personal interview, August and September, 2004.

2. Dr. Buffolino. Personal interview, November, 2004, and February, 2005.

3. Jeffreys, A.J., V. Wilson, and S.L. Thein. "Hypervariable 'Minisatellite' Regions in HumanDNA." Nature, 314(6006), 67–73, 1985.

4. Roewer, L., J. Arnemann, N.K. Spurr, K.H. Grzeschik, and J.T. Epplen. "Simple RepeatSequences on the Human Y Chromosome Are Equally Polymorphic as Their Autosomal Counterparts." Human Genetics, 89(4), 389–394, 1992.

5. Jobling, M.A., N. Bouzekri, and P.G. Taylor. "Hypervariable Digital DNA Codes for HumanPaternal Lineages: MVR-PCR at the Y-Specific Minisatellite, MSY1 (DYF155S1)." Human Molecular Genetics, 7(4), 643–653, 1998.

6. Jobling, M.A., A. Pandya, and C. Tyler-Smith. "The Y Chromosome in Forensic Analysis andPaternity Testing." International Journal of Legal Medicine, 110, 118–124, 1997.

7. Anderson, S., Bankier, A.T., Barrell, B.G., de Bruijn, M.H., Coulson, A.R., Drovin, J. et al."Sequence and Organization of the Human Mitochondrial Genome," Nature, 290(5806): 457–465, 1981.

8. Wilson, M.R., J.A. DiZinno, D. Polanskey, J. Replogle, and B. Budowle. "Validation ofMitochondrial DNA Sequencing for Forensic Casework Analysis." International Journal of Legal Medicine, 108(2), 74–86, 1995.

9. Grubwieser, P., A. Thaler, S. Köchl, R. Teissl, W. Rabl, and W. Parson. "Systematic Study onSTR Profiling Blood and Saliva Traces after Visualization of Fingerprint Marks." Journal of Forensic Science, 48(4), 733–741, 2003.

10. Raymond, J.J., C. Rouz, J. Sutton, and C. Lennard. "The Effect of Common FingerprintDetection Techniques on the DNA Typing of Fingerprints Deposited on Different Surfaces." Journal of Forensic Identification, 54(1), 22–44, 2004.

11. Geberth, V.J. Practical Homicide Investigation: Tactics, Procedures, and Forensic Techniques, 3rd ed. Boca Raton, FL: CRC Press, 1996.

12. Walsh, P.S., D.A. Metzger, and R. Higuchi. "Chelex 100 as a Medium for Simple Extraction ofDNA for PCR-Based Typing from Forensic Material." Biotechniques, 10(4), 506–513, 1991.

13. Gusamo, L., A. Gonzalez–Neira, C. Pestoni, M. Brion, M.V. Lareu, and A. Carracedo."Robustness of the Y STRs DYS19, DYS389 I and II, DYS390 and DYS393; Optimization of a PCR Pentplex." Forensic Science International, 106(3), 163–172, 1999.

14. Reynolds, R., G. Sensabaugh, and E. Blake. "Analysis of Genetic Markers in Forensic DNASamples Using the Polymerase Chain Reaction." Analytical Chemistry, 63(1), 2–15, 1991.

15. Chamberlain, J.S., R.A. Gibbs, J.E. Ranier, P.N. Nguyen, and C.T. Caskey. (1988). "DeletionScreening of the Duchenne Muscular Distrophy Locus via Multiplex DNA Amplification." Nucleic Acid Research, December, 16(23), 11141–11156, 1988.

16. Andrews, M., K. Iwona, P.F. Chinnery, R.N. Lightowlers, D.M. Turnbull, and N. Howell."Reanalysis and Revision of the Cambridge Reference Sequence for Human Mitochondria DNA". Nature Genetics, 23, 147, 1999.

17. Budowle, B., C. Ranajit, G. Carmody, and K.L. Monson. "Source Attribution of a ForensicDNA Profile." Forensic Science Communication, 2(3), 2000.

18. Frye v. United States, 293 F. 1013 (D.C. Cir. 1923).

19. Daubert v. Merrell Dow Pharmaceuticals, 509 US 579 (1993).

20. Brenner, C.H. "Symbolic Kinship Program." Genetics, 145, 535–542, 1997.

21. Prinz, M. and M. Sansone. "Y Chromosome-Specific Short Tandem Repeats in ForensicCasework." Croatian Medical Journal, 42(3), 288–291, 2001.

Selected Reading

Camp, F.R., Jr. "Forensic Serology in the United States. I. Blood Grouping and Blood Transfusion — Historical Aspects." American Journal of Forensic Medicine and Pathology, 1(1), 47–55, 1980.