by Mark Springer
On the morning of August 12th, a white Subaru Legacy GT was found, surrounded by yellow crime scene tape, in a parking lot at Applied Biosystems in Foster City, California. The hood of the car was open. A large amount of apparent blood was pooled in the hatchback area of the vehicle. Foul play? No, more like an innovative way to teach high school and college students about the use of DNA technology and other methods of analyzing evidence in the field of forensic science.
The creative culprit in this case was Mark Okuda, a biology teacher at Silver Creek High School in San Jose, California. His partner in mock crime: Frank Stephenson, Ph.D, Senior Technical Manager in the Technical Training Department at Applied Biosystems. The two educators set up this crime scene and together taught «Forensics PCR», a hands-on workshop jointly sponsored by Applied Biosystems and the Bay Area Biotechnology Education Consortium (BABEC), a non-profit network of six high school education outreach programs. After three days of intensive training in the use of state-of-the art tools of forensic science, 14 high school and college students were introduced to the expanding role of science and technology in criminal investigations.
«The class was actually better than I had hoped it would be,» said Kyle Siebenthall, a recent high school graduate, who is entering his freshman year at Cornell University.
«I didn’t expect that the first day that we came here that we would find a crime scene and a car,» he continued. «So, I was pretty impressed by that. It has been a thorough course. They’ve given us a lot of information and a lot of data to analyze. I get a kick out of working with the lab equipment.»
To Be a Crime Scene Investigator (CSI)
In their three-day walk in the shoes of a crime scene investigator (CSI), the students learned about forensic science by working in labs equipped with the same kind of high-tech equipment that is used by both modern-day criminalists and those who portray criminalists on television programs such as the top-rated TV show CSI.
BABEC actively promotes improvements in science classes in public and private high schools in San Mateo, Santa Clara, San Francisco, Marin, Alameda, and Contra Costa counties in the San Francisco, California area. Over 22,000 high school students, enrolled in biology, AP biology, life science, integrated science, and chemistry classes in 110 schools, participate in BABEC programs each year.
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At this BABEC-sponsored workshop at Applied Biosystems, the students performed the polymerase chain reaction (PCR) and amplified DNA samples on Applied Biosystems thermal cyclers. They quantified the amount of DNA in collected evidence samples using a special probe and performed a preliminary analysis to include or exclude suspects using the PCR and polyacrylamide gel electrophoresis. Finally, they analyzed the DNA of suspects by using the ABI PRISM® 3100 Genetic Analyzer and the AmpFLSTR® Identifiler® PCR Amplification Kit, sophisticated DNA analysis tools from Applied Biosystems. Some of these tools have also been featured on both the CSI and CSI: Miami television shows.
Just as television shows like CSI, Court TV, and Quincy feature criminal investigations that involve re-enactments of a crime, so too this workshop gave students the opportunity to play criminalists and re-enact their versions of the crime once they had gathered and analyzed evidence from a crime scene.
Collecting Physical Evidence
The workshop began with an early morning briefing on the case by Mr. Okuda. He described circumstantial evidence about the crime and distributed a series of evidence reports that included facts about DNA, ballistics, firearms, blood splatter analysis, and fiber evidence. The fledgling criminalists learned that the victim who was killed in this mock crime was named Erica Holmes, and the primary suspect was her boyfriend Marcus O’Neill.
According to a report filed by detective Gil Grissom (Okuda’s fictional choice of names based on the name of the investigator on CSI), Ms. Holmes had been working on the engine of her car when she was shot in the head. Later, her body was dumped into the San Francisco Bay. Six months after she had been reported missing by her parents, detective Grissom found her car at a San Francisco impoundment lot.
The students learned that Marcus O’Neill had failed a polygraph test in which he claimed no involvement in the death of Ms. Holmes. Later, however, he confessed that he was present during her death, but did not kill her. He claimed that, during a heated argument with Ms. Holmes, she attempted to attack him with a knife. In the scuffle that followed, she fatally stabbed herself in the throat. He didn’t think anyone would believe his story, so he dumped her body in the back of her car, and drove to a remote area of the San Francisco Bay, where he abandoned the vehicle. However, investigators obtained a search warrant and made a visit to O’Neill’s townhouse to examine the garage where O’Neill claimed that the fatal stabbing took place. On the garage floor, they found a cartridge case. In O’Neill’s work van, they found a handgun registered in his name. These findings seemed to cast some doubt on O’Neill’s explanation.
After surveying the crime scene, the students began to search for physical evidence that would either support or refute O’Neill’s questionable explanation of events.
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Forensic Science Solves Most Crimes
Short of a confession or an eyewitness account of a crime, examination of physical evidence, or forensic science, is what most frequently convicts suspects of a crime, and, according to Mr. Okuda, proves to be the way in which most crimes are solved.
Advances in technology have bolstered the abilities of criminalists to successfully tie physical evidence to criminal suspects. Perhaps the most significant of these technological advancements have been those associated with DNA fingerprinting techniques, which can be used to identify legitimate suspects and exonerate those who have been wrongfully accused of crimes.
However, technological advances in other areas of forensic science such as fiber analysis, histology, ballistics, and blood analysis now equip crime scene investigators with an arsenal of techniques for identifying criminal suspects, thereby making it even more difficult for criminals to elude justice.
Students had the opportunity to examine a wide variety of physical evidence from the crime scene. They peered at fiber samples under a microscope, and learned that based on certain telltale characteristics of different kinds of fibers, forensic scientists can decipher the nature of the fiber. For instance, scales on the outside of a fiber indicate it is a hair.
Histology also plays a big part in many crime investigations. For this investigation, students examined slides of tissue samples collected from the crime scene. The students learned that cells in these tissue samples that had shapes and colors characteristic of gyrus cells of the cerebral cortex provided evidence that trace amounts of brain tissue had been recovered from the engine bay of the Subaru.
For blood splatter analysis, students learned that a fine-mist splatter of blood comes from a high-velocity impact. Spots of blood may indicate that a bleeding body was carried away from the scene of a crime. The students also learned about the amazing properties of luminol, a chemical that investigators spray in suspicious areas of an apparently clean crime scene. When the lights are turned off, luminol emits a green glow wherever blood has been spilled. This glow, which results from a chemical reaction with blood proteins, can appear even if a criminal makes a determined effort to wipe away any trace of blood.
«What I have found is that you can get the kids attention if you relate [science] to something,» said Mr. Okuda, who also teaches a course in biotechnology and a forensics class. «The students really like the [forensics] course because it’s on TV a lot,» he said.
«Court TV has popularized [forensic science]. The students like [Okuda’s forensics course] because it’s a real-life application. After having biology, chemistry, and physics, they have a chance to apply these principles [learned in these classes] to a real-life situation. It’s a different kind of thinking because the answer is not going to be in the book,» noted Mr. Okuda.
This kind of hands-on learning appeals to Meghan Harrison, a senior at Salinas High School, who participated in the three-day workshop.
«What I liked about this class was that we didn’t just sit there and read it out of a book, because I don’t really learn a lot that way,» she says. «I like the way that they set up the crime scene and from that point step-by-step we did everything. When I came to this class, I really didn’t know a lot about this stuff, and I’ve learned so much.»
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The Life of a Criminalist
At its most intriguing, forensic science helps fictional characters like Columbo, Quincy, or Gil Grissom of CSI solve a crime. However, the real job of a crime scene investigator or a criminalist is a little less romantic than how it is often portrayed on television.
As part of the workshop, Cordellia Willis, a criminalist for the Santa Clara County Crime Lab, and Catherine Caballero, a former criminalist, and now a field application specialist for Applied Biosystems—who also trains Applied Biosystems employees and customers in forensic science—spoke to the students about their experiences as criminalists. They gave the students a glimpse of the life of a modern-day crime scene investigator, making clear distinctions between the job responsibilities carried out by television show criminalists and the actual duties of a professional.
For instance, unlike television show criminalists, who often also do the work of a detective, Ms. Willis, who serves the police departments of San Jose, Palo Alto, and all of Santa Clara county, never participates in questioning suspects about presumed guilt or innocence. In fact, criminalists never solve a case. Instead, they supply evidence that can be used in a court of law to ultimately convict a suspect of a crime. Other than testifying in court, criminalists do all of their work behind the scenes, most of it in the lab.
Even so, criminalists do benefit from possessing the skills of a detective. For instance, like detectives, criminalists must be vigilant, knowing where all the evidence for a crime has been at all times. They keep a record of the «chain of custody,» an account of all the people who have handled the evidence for a crime throughout the course of an investigation. As Ms. Willis noted, the criminalist tries to keep that number of people to a minimum, especially when handling DNA samples collected from a crime scene.
«Contamination of samples is always a risk. We are always shedding our DNA in our skin cells, hair, and sweat,» noted Ms. Willis.
To better understand some of the regulations and restrictions that a criminalist faces when examining DNA samples, students in the class were advised to not talk in the lab. DNA from their sprayed saliva could potentially contaminate the DNA samples that they were analyzing.
DNA Has Changed the Face of Forensic Science
DNA, and related technologies for analyzing nucleic acids, have dramatically changed the way in which forensic scientists approach a criminal investigation. Several characteristics of DNA make it an ideal marker for identifying individuals who may have been present during the commission of a crime.
For one, DNA is a stable molecule, so that, if it is shed at a crime scene, it stays around for awhile. Also, DNA does not easily fall apart during collection or analysis techniques. Furthermore, DNA has a high discrimination power, so it can be used to identify individuals. A double-stranded molecule, each strand of DNA is a string of bases or DNA letters (A, T, C, and G) that form patterns unique to an individual in different stretches of a strand. Criminalists use these patterns like fingerprints to identify different individuals. Moreover, DNA is prevalent, found in all nucleated cells.
According to Ms. Willis, DNA can be obtained from many different sources. It can be extracted from samples of blood, semen, saliva, hair, roots, bone, teeth, tissue, skin, and sweat. It has been collected from such odd places as chewing gum, envelope flaps, balloons, toothbrushes, dandruff, cigarette butts, bottles and cans, or tape ripped by a suspect’s teeth in the commission of a crime. Criminalists have even lifted DNA from bits of food left at the scene of a crime by an absent-minded criminal, who grabs a bite to eat and leaves some of his snack behind. Unfortunately for criminalists, the prevalence of DNA means that sometimes they have the unsavory job of extracting it from such distasteful sources as vomit and dirty diapers.
Another advantage of working with DNA is that it is possible to perform a differential extraction and separate sperm DNA from skin DNA, or an individual’s blood sample from within a sample of mixed blood from multiple individuals.
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How to See DNA
Just as important as the characteristics of DNA that make it a valuable marker for crime scene investigations, are the advances in technologies that have made it possible for forensic scientists to detect molecular markers within DNA and match those markers to individuals who possess them.
Restriction fragment length polymorphisms (RFLPs) were the first DNA markers used in forensic science. RFLPs are sets of DNA fragments of varying lengths created by restriction enzymes, DNA-cutting enzymes harvested from bacteria and used as biological scissors to cut DNA between specific base pairs. Polymorphisms, variations in base sequences that occur at cutting sites throughout DNA, result in the same restriction enzyme cutting DNA at different places for different individuals, creating sets of DNA fragments of different lengths, or a characteristic collection of fragment lengths based on the DNA base-pair sequence of an individual. One of the biggest drawbacks to RFLPs, however, is the length of time required for sample analysis—up to seven weeks after fragment cuttings are done to create a pattern that identifies an individual.
According to Ms. Willis, a real boon for identification by DNA came about in the early 1990s with the introduction of short tandem repeat (STR) analysis, a polymerase chain reaction (PCR)-based DNA analysis method that requires just a small amount of genetic material, and a short analysis time—days instead of weeks. Moreover, the integrity of a DNA sample matters less with STRs than with RFLPs, so criminalists are better able to identify degraded DNA using STRs. With the quantity and integrity of the DNA sample being less important with PCR-based typing methods than with conventional RFLP methods, STRs have become the markers of choice for most DNA fingerprinting efforts.
STRs are repeating sequences of two to five bases that lie adjacent to each other in different regions throughout genomic DNA. STRs are very abundant in the human genome. In STR analysis, an investigator analyzes a DNA sample by examining several different regions, or loci of the DNA, for the number of times a particular pattern of bases is repeated in tandem units. An allelic ladder shows all possible numbers of STRs at each interrogated loci.
Criminalists use software that depicts STRs as peaks on a graphical display. Overlaying peak data for an unknown sample with an allelic ladder reveals how many copies of an STR exist at each locus for each chromosome (one from each parent) of the unknown sample. For example, a person who has four copies of an STR at one location on one chromosome and three copies of the same STR at a corresponding locus on the other chromosome, would have a genotype of 3,4 at that locus. A genotype of 4,4, has the same number of STRs on each chromosome and is referred to as homozygous.
Data derived from an STR analysis can be used to determine the father of a child in paternity cases, or in forensic cases such as this mock crime case, the identity of a suspect can be determined by matching STR patterns in a donated DNA sample to those in unknown DNA extracted from a blood stain found at the crime scene.
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Students learned that all crime labs must first quantify the amount of DNA found at a crime scene before they can analyze it. But, instead of just telling them this, Dr. Stephenson led the budding crime sleuths to the lab where they used the QuantiBlot® Human DNA Quantitation Kit from Applied Biosystems.
The QuantiBlot kit exploits the complementary base-pairing of DNA to quantify how much DNA is present in a sample. To do this, the kit uses, as a probe, a piece of DNA that matches a targeted stretch of complementary base pairs. The probe attaches itself to a target DNA region—the centromere of chromosome 17—in a manner similar to the way that two pieces of Velcro stick to one another. The probe is biotinylated—labeled with a biotin molecule—so that scientist can visualize and quantify the targeted DNA by using chemiluminescent detection.
The QuantiBlot kit can be used not only to determine how much DNA there is in a sample, but also to determine if the DNA came from a primate—such as a chimpanzee, a gorilla, or a human.
In this mock crime scene investigation, use of the QuantiBlot kit allowed the novice criminalists to home in on the source of the blood found in the cargo bay of the hatchback. If the probe sticks, it shows that the blood came from a primate, not from an animal such as a deer or a pig.
A positive test for primate blood eliminates the possibility that the blood in the car came as the result of a hunting expedition or the slaughtering of an animal, but instead it came from a primate, presumably a human.
STR Analysis in the Lab
Frank Stephenson taught the students how to perform all of the steps in an STR analysis of DNA samples that supposedly had been collected from under the hood and inside the hatchback of Erica Holmes’s vehicle, and from Marcus O’Neill, and from the parents of Erica Holmes: Sarah and Dwayne Holmes. Of course, in order to perform the actual analysis, the students had to have real DNA samples, which were graciously donated by Dr. Stephenson and members of his family.
The first step in performing an STR analysis is to amplify the DNA in a sample and label the STRs with a fluorescent dye, so that STR patterns can be visualized. Regions of DNA containing STRs are simultaneously copied and labeled during the PCR. To perform the PCR, the students place their samples in an Applied Biosystems thermal cycler, an instrument that repeats cycles of heating and cooling according to a pre-programmed protocol. Heating DNA separates the two complementary strands of DNA. Cooling the DNA enables fluorescently labeled probes to anneal to STRs at specified regions in a single strand of DNA. To label the fragments, the students used the AmpFLSTR® Identifiler® PCR Amplification Kit, a reagent kit that can be used to amplify and label sixteen different loci in a DNA sample with fluorescent dyes.
Once DNA samples are amplified, scientists characterize the STRs by polyacrylamide gel or capillary electrophoresis methods. These nucleic acid separation techniques are also used for DNA sequencing applications. Students in the workshop used the ABI PRISM 3100 Genetic Analyzer, an automated capillary electrophoresis instrument that uses hair-thin capillary tubes to separate dye-labeled DNA fragments containing STRs. The 3100 Genetic Analyzer then uses a laser to stimulate fluorescent emission from the dyes. A charge-coupled device (CCD) camera detects dye-labeled DNA fragments, and the system determines their quantities and sizes by using an internal size standard.
The software packaged with the AmpFLSTR kit estimates the size of different DNA fragments and correlates the size of a fragment with a particular number of STRs in a locus.
Applied Biosystems provides a number of different kits for amplifying and labeling STR loci. As Catherine Caballero notes, «The only difference between one kit and the next is the region of DNA that we’re looking at.»
As for the ease of use of the high-tech equipment, Mr. Okuda remarked about his students, «They pick it up sooner than I do. For example, they found the computer programs on the thermal cycler are very easy to use.»
Both the 3100 Genetic Analyzer and the AmpFLSTR kits have also helped criminalists populate computer databases with DNA sample data. These databases can then be searched for matching base sequences when DNA samples of unknown origin are collected at crime scenes. «So, some of these databasing labs need high throughput and they need automation,» says Ms. Caballero.
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CODIS Captures Suspects
The success of STR analysis in identifying crime suspects has prompted the Federal Bureau of Investigation (FBI) of the United States to select 13 STR loci to constitute the core of a national database of DNA markers known as the Combined DNA Index System (CODIS). Law enforcement officials hope one day to be able to identify perpetrators of violent crimes by accessing their DNA fingerprints in a national computer. One of the challenges still facing proponents of a national database of DNA samples is that every state has different restrictions that place limits on the collection of DNA. At present, a version of the CODIS database exists, but is not yet fully operational. Criminalists will also benefit from access to CODIS because they will be able to search databases for a genetic profile that matches DNA evidence collected at a crime scene.
Mitochondrial DNA Can Be Used to Identify Human Remains
Although STR analysis provides criminalists with a powerful technique for determining if someone was present at a crime scene, sometimes a forensic scientist must take a less direct DNA route to identifying someone. For example, in the mock crime, the only evidence of the body of Erica Holmes was a femur bone found in the San Francisco Bay by a fisherman. The situation was complicated by a report from a woman named Myla Graystone. She said that her sister Rita had disappeared several months earlier in the same vicinity where the bone was recovered. In order to positively identify the bone, the novice criminalists turned to mitochondrial DNA (mtDNA) analysis.
When attempting to confirm the identify of an unknown individual from remains such as bones or other body parts, forensic scientists analyze mtDNA for a match between the DNA of the remains and that of a maternal relative of a person whose remains they believe that they have found.
«When trying to recover DNA from old tissue samples and bone, you might be successful in recovering mtDNA, but not genomic DNA, particularly in a case where you have burnt tissue,» Dr. Stephenson noted.
Mitochondrial DNA comes from the mitochondria, the powerhouses of our cells. Mitochondria manufacture ATP and are present in almost every cell.
Mitochondrial DNA can be used to determine maternal heritage because, at conception, mitochondria contained within the egg of a female are passed almost exclusively from the mother to the offspring. The mitochondria of the sperm are at the base of its tail. According to Dr. Stephenson, «When the sperm fertilizes the egg, it leaves behind the tail, along with all the energy-producing mitochondria. Only rarely will mitochondria from male sperm reach the egg.»
Mitochondrial DNA can survive in ancient bone specimens, degraded body parts, or hair. Mitochondrial DNA stays intact under conditions in which nuclear DNA degrades. Even in harsh conditions such as fire or prolonged decomposition, mtDNA maintains its genetic structure. One reason that researchers have a greater likelihood of finding intact mtDNA than they would of finding nuclear DNA in ancient specimens is that there is so much more mtDNA than nuclear DNA in cells. A single cell may have hundreds to thousands of copies of mtDNA, but cells have just two copies of nuclear DNA. The hardiness of this kind of DNA also makes it useful for identifying individuals from ancient remains.
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Also, mtDNA is short-only about 16,000 base pairs long-and it’s circular, so there is less of a chance that mtDNA will break than nuclear DNA.
«Circular DNA tends to be more stable than linear DNA,» explains Dr. Stephenson.
Mitochondrial DNA analysis that has been performed with Applied Biosystems technology has helped scientists to identify ancient human remains and unravel a number of historical and personal mysteries.
For example, mtDNA analysis was used to positively identify the remains of Tsar Nicholas II, resolving a long-standing controversy surrounding the murdered last tsar and his family.
In 1998, mtDNA analysis identified Michael Blassie as the solider whose remains had laid unidentified in the Tomb of the Unknowns at Arlington National Cemetery for 14 years.
In December 1999, scientists solved one of the great mysteries of European history by using mtDNA analysis to prove that the son of executed French King Louis XVI and Marie-Antoinette died as a child in prison.
«I’d say between 80 to 85 percent of all human identification work is done with Applied Biosystems human identification chemistry and software kits,» noted Ms. Caballero.
While human identification from ancient remains may have personal and historical significance, natural and man-made disasters represent the most frequent need to identify humans based on DNA recovered from their remains. For example, the September 11th attacks on the World Trade Centers in New York have resulted in the world’s largest DNA-based identification effort from a single incident in the history of forensic science. To help identify vicims of this tragedy, Applied Biosystems and Celera Genomics are now conducting mtDNA sequence analysis of extracted DNA from family reference specimens and evidentiary material from the disaster site.
Once scientists extract mtDNA from bone, they then perform the PCR to amplify a region of the DNA that has patterns of bases unique to a maternal line of heritage.
«In the case of our crime, we have a bone recovered from the San Francisco Bay. It might be from Rita Graystone, or from Erica Holmes,» Mr. Okuda said.
When comparing mtDNA of remains with family DNA samples, scientists examine a region of mtDNA bases that can withstand mutations. Sequences of DNA bases that do not code for peptides involved in essential roles in the manufacturing of energy for the cell vary between individuals. For that reason, forensic scientists select primers and probes that will amplify a non-coding 1,210 base-pair region of mtDNA known as the control region.
In a comparison between mtDNA recovered from the femur bone recovered from the Bay, and samples donated by Myla Graystone (the sister of Rita), and by the mother of Erica, the students discovered that the mtDNA from the bone matched that of Erica’s mother, strongly suggesting that the remains recovered from the San Francisco Bay were those of Erica Holmes.
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After teams of three to four students each pieced together a puzzle of physical clues left behind at the crime scene, they made a short presentation to the group, re-enacting what they believed had happened on the day Erica Holmes was killed.
Although all of the groups presented overwhelming evidence that Marcus O’Neill murdered his girlfriend, Erica Holmes, the versions of how this happened varied. However, for the students, perhaps of greater significance than finding out exactly what happened on that fictitious day was the experience of using the same kind of equipment that CSIs use, and learning how CSIs do their jobs.
«I think that one of the things that attracts [the students] is that the high-tech equipment that they use in my class are the same machines that they might see a scientist using on TV, so it empowers them to say, ‘Hey, I can do these things,’ and it may motivate them to pursue a career related to molecular biology,» said Mr. Okuda.
However, some of the grisly realities of working with corpses make some people re-evaluate choosing a career as a crime scene investigator.
«A lot of people get out because they just can’t stand constantly seeing ugliness every day,» said Ms. Caballero.
Or, as Brenton Hughes of Gunn High School in Palo Alto noted at the end of the three-day workshop:
«It’s [forensic science] a lot nastier than CSI,» he said.
Still, some students, like Veronica Espinoza, James Logan High School, were inspired about the possibilities of pursuing a career in forensic science:
«This workshop has made me consider a career in forensic science as an option after going to college,» she said.
by Mark Springer