Article Curriculum and Course Materials for a Forensic DNA Biology Course

Kelly M. Elkins*

*From the Department of Chemistry, Towson University, 8000 York Road, Towson, Maryland 21252

Abstract The Forensic Science Education Programs Accreditation Commission (FEPAC) requires accredited programs offer a “coherent curriculum” to ensure each student gains a “thorough grounding of the natural. . .sciences.” Part of this curriculum includes completion of a minimum of 15 semester-hours forensic science coursework, nine of which can involve a class in forensic DNA biology. Departments that have obtained or are pursuing FEPAC accreditation can meet this requirement by offering a stand-alone forensic DNA biology course; however, materials necessary to instruct students are often homegrown and not standar-

dized; in addition, until recently, the community lacked commercially available books, lab manuals, and teaching materials, and many of the best pedagogical resources were scattered across various peer-reviewed journals. The curriculum discussed below is an attempt to synthesize this disparate information, and although certainly not the only acceptable methodology, the below discussion represents “a way” for synthesizing and aggregating this inforC 2013 by mation into a cohesive, comprehensive whole. V The International Union of Biochemistry and Molecular Biology, 42(1):15–28, 2014

Keywords: curriculum design development and implementation; molecular Biology; computers in research and teaching; genomics proteomics bioinformatics; new course development; nucleic acid structure function and processing; biotechnology education

Introduction The Forensic Science Education Programs Accreditation Commission (FEPAC) requires accredited programs offer a “coherent curriculum” to ensure each student gains a “thorough grounding of the natural…sciences” (Forensic Science Education Programs Accreditation Commission Accreditation Standards, revised November 9, 2012). Part of this curriculum includes completion of a minimum of 15 semester-hours forensic science coursework, nine of which can involve a class in forensic DNA biology. Departments that have obtained or are pursuing FEPAC accreditation can meet this requirement by offering a stand-alone forensic DNA biology course; however, materials necessary to instruct students are often homegrown and not standar-

dized; in addition, until recently, the community lacked commercially available books, lab manuals, and teaching materials, and many of the best pedagogical resources were contained in various peer-reviewed journals. For example, some recent papers detail new laboratory experiments focused on forensic DNA analysis in the undergraduate forensic laboratory and biochemistry laboratory that can be adapted for use in the undergraduate forensic DNA biology course. The curriculum discussed below is an attempt to synthesize this disparate information, and although certainly not the only acceptable methodology, the below discussion represents “a way” for synthesizing and aggregating this information into a cohesive, comprehensive whole.

Sample Courses *Address for correspondence to: Assistant Professor of Chemistry, Department of Chemistry, Towson University, 8000 York Road, Towson, Maryland 21252. E-mail: [email protected] Received 31 May 2013; Accepted 25 September 2013 DOI 10.1002/bmb.20749 Published online in Wiley Online Library (wileyonlinelibrary.com)

Biochemistry and Molecular Biology Education

This project originally developed from the author’s efforts to develop the curriculum for the revised Criminalistics II course at the Metropolitan State College (now University) of Denver (Metro State) in the spring of 2008. Criminalistics II (CHE 3710), is a forensic DNA biology 4-credit, one-semester, lecture plus laboratory upper-division undergraduate course. Each lecture and lab meets weekly for 3 hours. The

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Biochemistry and Molecular Biology Education class has been taught annually since its inception to class sizes of eight to 15 students. The students are assessed on their performance based on three to four monthly in-class exams, one final exam, 15 weekly laboratory reports, weekly quizzes based on the lab material, a short research paper on a case reported in the primary literature, and an oral presentation based on the paper. A variant of this course, Forensic Biology (CHEM 461), will be taught at Towson University during the fall semester of 2013. This 3-credit, one semester, upper-division undergraduate course will meet weekly for 3 hours each, with laboratory exercises integrated into the lectures. Students will be assessed using a combination of exams, laboratory write-ups and a research paper. Both Metro State and Towson University are FEPACaccredited programs, and each offers FEPAC-accredited undergraduate programs in forensic chemistry. Towson University also offers a Master of Science in Forensic Science (MSFS) degree. While the study of forensic DNA biology has long been an important topic in several courses of study by students pursuing the MSFS at Towson, there has been no stand-alone, undergraduate curriculum in forensic biology until now. Both courses have a pre-requisite of a full-year of organic chemistry and labs. Both are typically taken after a forensic chemistry course and courses in general biology, genetics, and statistics.

Pre-requisites and Related Courses In addition to coursework in analytical and organic chemistry and instrument analysis, FEPAC accreditation requires students to complete coursework in biochemistry, molecular biology, genetics, ethics, calculus, physics, and statistics. A stand-alone, upper-level offering in forensic DNA biology can integrate much of the theoretical underpinnings of these other, more generalized courses. In addition, a standalone curriculum also offers the opportunity to introduce students to what’s unique about forensic DNA biology, including best practices for evidence collection, issues surrounding contamination in the lab and during field work, and legal issues. Forensic biology courses vary in emphasis, in part due to the department (e.g., chemistry, biology, or criminal justice), but other factors such as time, budget, and instructor expertise influence how the course is taught. Most of the students who enroll in forensic biology courses are chemistry or biology majors pursuing a degree in those fields with a concentration in criminalistics or forensic science, or are pursuing a specialized degree in forensic biology or forensic chemistry. However, both of these two universities have allowed upper-level chemistry and biology students that meet most of the pre-requisites to enroll. At this time, a statistics class is not a pre-requisite for the course; however, students are recommended to have completed statis-

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tics prior to enrolling in the course due to the importance of this topic for DNA molecular biology.

Course Topics The main goals of courses in forensic biology are to introduce students to modern DNA typing, laboratory practices, the applicability of DNA to criminalistics, and to challenge students to think critically about new methodologies and the future of the profession. The emphasis of the laboratory is to prepare students to work with biological evidence, perform standard assays and DNA typing, interpret results, and perform statistical analyses. A special emphasis is placed on evaluating imperfect scenarios including degraded, low template, and mixture samples as encountered in crime lab work.

Course Materials As with any course, the decisions regarding which books and course materials to adopt depend upon the focus of the particular course, the instructor’s preferences, and the quality of the materials. Fortunately, there are now several excellent resources available for consideration. For example, CHEM 3710 is focused on human body fluids analysis and DNA typing in criminalistics. Between 2008 and 2011, this course used Forensic DNA Typing: Biology, Technology and Genetics of STR Markers, published by John Butler in 2005 [1]. At the time this book was adopted for the course, there were no suitable alternatives. Updated books were released in 2010 and 2011, including Advanced Topics in Forensic DNA Typing: Methodology published in 2011 which was used for the Spring and Fall 2012 course offerings [2, 3]. John Butler is also the author of the first book focused on the subject, Forensic DNA Typing: Biology & Technology Behind STR Markers, which was published in 2001 [4]. CHEM 3710 was supplemented with ancillary materials (e.g. World Wide Web resources and primary literature) to cover topics such as natural fiber microscopy and presumptive testing of body fluids. For example, lectures on hair analysis are based on two 2004 articles published in Forensic Science Communications by Deedrick and Koch [5, 6]. Virkler and Lednev’s excellent review of presumptive tests for biological materials is used in teaching serology and presumptive tests [7]. Although Butler’s texts are geared as a reference for practitioners rather than as a textbook, they are still a good choice for this course due to the depth of information they provide. Additionally, many of his figures and sample data are available on STRBase (www.cstl.nist.gov/strbase) in Microsoft PowerPointV format. PowerPoint lectures of each chapter in the selected books were created for use in the classes. Li’s Forensic Biology published in 2008 is another book on the subject and has been adopted for R

Curriculum and Course Materials

CHEM 461 due to its emphasis on body fluids analysis and presumptive and screening tests [8]. CHEM 3710 and CHEM 461 are focused on human DNA typing and body fluids analysis; however, the focus of forensic DNA courses can be broadened to include DNA typing of any DNA-containing organism. These topics are covered by books including Microbial Forensics by Budowle et al. [9], Forensic Entomology by Gennard [10], and Fundamentals of Forensic Anthropology by Klepinger [11] and others.

Lecture Topics This curriculum (Table I) focuses on introducing students to modern human DNA typing, laboratory practices, ethics, and the applicability of DNA to criminalistics. In the first unit, students are introduced to the history of forensic DNA biology through the analysis of various high-profile cases, including the Jeffreys, Lewinsky, and O.J. Simpson cases. I also briefly cover a historical perspective of human identification beginning with blood typing and isozyme protein electrophoresis assays through the modern minisatellite short tandem repeat (STR), mitochondrial DNA (mtDNA) sequence analysis, and single nucleotide polymorphism (SNP) techniques. For example, students analyze these methods using a number of criteria, including their cost and speed, discriminating power, ease of use, minimum length of DNA assayed, minimum amount of DNA needed, condition of DNA required, form of DNA used, as well as the potential of automation, multiplexing, and data processing [1, 3]. Such an analysis helps students to understand the development of the field from an historical perspective, and provides a solid context for how and why the field has advanced that way it has. Although general biology is a pre-requisite for this course, we also briefly review DNA biology fundamentals in order to ensure all students begin the class with a common understanding of the material. The course emphasizes the structures of the nucleotide bases and nucleic acids, location of DNA in the cell, which cells lack DNA, copy number, Watson–Crick base-pairing, DNA melting and melting temperature, denaturation and renaturation, replication/amplification, DNA degradation in vitro and in vivo, and sizes of representative genomes. Students are exposed to principles of heredity and discrimination challenges, including cases in which individuals have the same nuclear DNA profile (i.e. identical twins) that are a result of biological underpinnings of a single fertilized cell splitting. This last concept is particularly important, since discrimination challenges are central to both technological and legal-politico issues many students will face as professional forensic biologists. Students are next introduced to basic crime scene investigation techniques, sample collection and storage protocols, and presumptive testing techniques of biological fluids and materials. These topics are normally not taught in

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first-year undergraduate biology and chemistry courses and must be taught to the students so that they have the background and context for the application to criminalistics. Presumptive testing topics covered include chemical, instrumental, and microscopic techniques for locating body fluids and hair evidence, as well as methodologies for tentative attribution of hair and body fluid samples as human origin [7]. Hair morphology, natural fiber differentiation, and techniques for determining the originating body location of hairs using comparative microscopy is taught using the methodologies outlined by Deedrick and Koch [5, 6]. In the second unit, students are introduced to modern human DNA typing including DNA extraction methods, DNA quantitation methods and PCR, common STRs, STR DNA typing methods and DNA separation technologies. DNA extraction methods including the phenol–chloroform– isoamyl alcohol, Chelex, dialysis, FTA paper, and methods employing commercial kits are introduced. The students are taught to evaluate the reagents for their chemical use and to determine the purpose of each reagent in order to evaluate the comparative effectiveness of various novel methodologies described in the peer-reviewed literature. The importance and use of reference, elimination, and control samples to compare to evidence samples is discussed in terms of ensuring the methods are working properly and there are no contamination issues. The polymerase chain reaction (PCR) and real-time PCR techniques used to amplify DNA in a test tube from extremely small DNA evidence samples are introduced. Topics include primer design, multiplexing, necessary reagents, “hot start,” thermocyling, DNA detection methods (e.g. radioactive labels, silver stain, fluorescent dyes and intercalating agents), PCR inhibitors, and the importance of clean technique. Students are introduced to DNA quantification techniques including UV–vis spectroscopy, fluorescence spectroscopy, agarose gel electrophoresis, real-time PCR assays, slot blot assays, end point PCR, the AluQuant system, Plexor HY, Quantifiler and PicoGreen microtiter plate assays. The TaqMan PCR assay used in the Quantifiler DNA quantitation kit is also introduced. Separation theories including Ogston sieving and reptation and denaturing and native gel electrophoresis are also discussed [1, 3, 11]. Students are next introduced the commonly used STR markers in the Combined DNA Index System (CODIS) database, the potential future CODIS markers, and desirable characteristics for STRs. Students are taught how to access DNA loci using GeneBank, and to interpret chromosome location, type of repeat, length of repeat, number of repeats, and naming conventions. These skills are essential for them if they encounter non-human or other DNA for which they need to develop a new assay to perform DNA typing. The use and implementation of commercially available STR multiplex kits (e.g. PowerPlex16 and AmpFlSTR) and miniSTR kits (e.g. miniFiler) is presented. Students learn how STR lengths and fluorescent probes for STRs can

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TABLE I

Forensic DNA biology lecture and laboratory course objectives synchronization matrix for an academic semester

Course objectives

Lab

Week 1 Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

History DNA typing DNA Biology review

NAa

Understanding of CSI Understanding of basic laboratory safety

1 UNIT 1

Sample collection best practices Microscopy of natural fibers Proper storage and characterization methodologies

2

3

Serology DNA extraction methodologies and techniques

4

DNA quantitation methodologies and techniques

5, 6, 8

Polymerase chain reaction

8, 9, 10

Biology Issues to Amplification & Analysis

8, 9, 10

UNIT 2

Commonly used short tandem repeat (STR) markers DNA Separation Methods

11, 12

STR DNA typing analysis Week 8 Week 9 Week 10 Week 11 Week 12 Week 13 Week 14 Week 15 Statistics and probability Commonly used databases Mixture analysis

13 Handout

Missing Persons and Disaster Victim Identification Efforts; Paternity & Maternity

18

Degraded DNA issues

19

Low-Level DNA Testing: Issues, Concerns, Solutions

19

SNPs

14

UNIT 3

Y chromosome DNA typing

N/Aa

Mitochondrial DNA analysis (mtDNA)

14

X-Chromosome DNA typing Non-Human DNA Testing New Technologies, Automation, and Software

N/Aa

UNIT 4

15

Proficiency Testing Lab accreditation

Case study

Ethics, Law, and the Scientific Expert in the Court a

These topics are covered in the laboratory manual but there is not sufficient time in the course to perform those lab experiments.

vary between kits. This is essential as the allele ladders vary accordingly among kits and are not interchangeable. DNA separation methods such as how to run a capillary electrophoresis (CE) instrument and fragment electrophoresis gels are presented, which are essential to DNA typing. Both capillary and slab gel electrophoresis are discussed including the respective Applied Biosystems (e.g. 310, 377, 3100, 3100, 3100-Avant, 3700, and 3730) and the Hitachi (e.g. FMBIO) instruments and modern next generation sequencing instruments. These techniques are compared and contrasted in terms of reagents, use, highthroughput capability, number of fluorescent dyes, automation, voltage used, time per sample run, quantity of sample required, data processing and visualization, and cost—all essential criteria to be considered by labs with real resource constraints when deciding which instrument type to purchase. DNA detection using multiple fluorescent dye labels is emphasized [1, 3]. This modern DNA labeling method varies in its implementation as commercial kits employ four or five dyes. DNA fragment analysis from the electropherogram or gel results is emphasized to obtain the DNA profile. The functions of the internal standard, ladder, and color matrix are introduced and students are given hands-on experience with peak-picking and performing allele calls. These are important for interpreting the correct allele call. Students are introduced to the complexities of STR amplification and analysis in terms of biology issues including degraded DNA and concentration-dependent effects, detecting strand slippage and stutter products, split peaks and poly-A addition, low copy number, allele dropout, sample mixtures, non-template addition, sample degradation, heterozygous peak imbalance, PCR inhibition, and contamination that may require that samples need to be repeated. Rarer events including tri-allelic patterns, microvariants, off-ladder alleles, null alleles, chromosomal abnormalities, extrachromosomal fragments, genetic chimerism, mutation, and artifacts are introduced. Students also learn about troubleshooting the CE instrument including how to overcome pull-up/matrix failure, signal drop-off, dye blobs, air bubbles, contamination, urea crystals, voltage spikes, and smiling and how to calibrate the instrument for dye processing and the used of an internal size standard. Instrument validation and the use of CE in DNA sequencing (e.g. for mitochondrial DNA typing) is also discussed [1, 3, 11]. Although previous coursework in statistics and probability is not a pre-requisite for this course, a thorough understanding of these concepts is critical to discriminating between two samples. As noted by the Scientific Working Group on DNA Analysis Methods (SWGDAM) (http://www. fbi.gov/about-us/lab/biometric-analysis/codis/swgdam.pdf, accessed July 11, 2013), DNA analysts must verify DNA typing results derived through analysis of analytical software. Consequently, this curriculum devotes the bulk of unit three to the study of statistics and probability, creating and using

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DNA databases, mixture analysis, low template and degraded DNA, cases of missing persons, and disputed maternity and disputed paternity. Students are introduced to integral genetics concepts including Mendelian genetics, Hardy-Weinberg equilibrium statistics and probability, the product rule, as well as error and concepts of population genetics such as, random sampling, linkage, and principles of diversity. Students are taught how DNA profile databases are assembled and how allele frequency and minimum allele frequency estimates for rare alleles are determined. These databases are essential to statistical calculations performed by crime laboratories. Students learn to estimate the profile frequency and how to use correction factors, as well as how to use databases to search for a match. Discriminating power, exclusion power, power of identity, random match probability, paternity index, and probability of paternity exclusion are also discussed. Students learn statistical approaches for the analysis of mixtures and partial DNA profiles/degraded DNA including the combined probability of exclusion and likelihood ratio methods for treating mixtures. Students are also introduced to Bayes’ theorem and source attribution. Kinship (reverse parentage) and parentage testing (often paternity testing) and statistical calculations used to determine the strength of a match are discussed. These cases are common in the legal system. DNA testing in mass disaster victim identification and immigration are also discussed [1, 3, 11]. Some other excellent books that cover the relevant statistics include A Primer of Population Genetics by Hartl [13], Statistical DNA Forensics: Theory, Methods and Computation by Fung and Hu [14], and An Introduction to Forensic Genetics by Goodwin et al. [15]. Finally, unit four introduces students to advanced topics, including more complex methods of human DNA typing using the X, Y, and mitochondrial chromosomes. Y-chromosome structure and DNA testing including Y-STRs and biallelic markers (e.g. AMEL, SNPs, and Y-Alu) and discriminating power is discussed. X-chromosomal structure and their use in DNA typing and lineage analysis, as well as commercial X- and Y-chromosome profile kits are introduced. Students learn about methods of mitochondrial DNA analysis including sequencing and using SNPs in lineage analyses. Non-human DNA testing (e.g. domestic and wild animals, microbial, viral, and fungal forensics) sources, cases, and challenges are discussed [1, 3]. New technologies including SNP typing assays, the reverse dot blot, genetic bit analysis, direct sequencing, denaturing HPLC, TaqMan assay, mass spectrometry, fluorescence polarization, high density arrays, electronic dot blot, molecular beacons, oligonucleotide ligation assay, melting temperature shift genotyping, pyrosequencing, minisequencing, allele-specific hybridization, and SNPstream UHT are also discussed. These are abundant in the literature and scientists must continually review whether their current methods are still the best for

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Biochemistry and Molecular Biology Education answering their desired questions. New automation and technologies including the “lab-on-a-chip,” hybridization assay, microchip CE, and MALDI-TOF implementation are also discussed [1, 3]. The course closes with a study of the legal aspects of DNA testing and the role of the scientific expert in a court of law, including the role of advanced instruments, methods validation, the three Rs (robust, reliable, reproducible), lab accreditation and proficiency testing. Forensic science and crime laboratories may be accredited or seeking accreditation; as such analysts need to be familiar with the process. Topics include quality assurance (QA), quality control (QC), “blind” testing, standard reference materials, and audits. These tests are commonly given to lab analysts in DNA laboratories. In addition to the science of DNA typing, students learn about the laboratory structure, governing bodies and review boards. Students are introduced to organizations such as the Technical Working Group on DNA Analysis Methods (TWGDAM), the SWGDAM, the DNA Advisory Board (DAB), the American Society of Crime Laboratory Directors (ASCLD), the National Institute of Standards and Technology (NIST), and the European Network of Forensic Science Institutes (ENFSI) among others that have worked to standardize and improve the field. Students learn about DNA Databases in Forensic Science (e.g. public NCBI and STRbase at NIST since 1997 [16] and private FBI CODIS) and what constitutes a “hit,” the cost of use, how data is submitted and scrubbed, how data can be purged, and criteria for system access [1, 3]. At the end of each course, students write a research paper on a topic or case study in forensic DNA biology from the literature and present short presentation on their project.

Published Laboratory Experiments Adaptable to Forensic Science When the first forensic science courses were taught beginning in the 1970s, the use of DNA as forensic evidence was uncommon because methods for analyzing this material had not yet been developed. However, much of the current literature is focused on DNA, especially on laboratory experiments. And although most of these experiments are written for the biochemistry or molecular biology laboratory, they provide forensic scientists with excellent ideas that can be adapted to the forensic biology laboratory. For example, in the Journal of Chemical Education, papers detail DNA typing of the human D1S80 locus implemented as an undergraduate biochemistry laboratory experiment [17], using mitochondrial DNA to probe a hypervariable region from simulated forensic samples in the biochemistry laboratory [18], forensic analysis of canine DNA samples from dog hair and saliva in the biochemistry laboratory [19], probing for a DNA segment present in genetically modified foods [20], genotype the normal varia-

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tion in human color vision [21], evaluate a metabolic polymorphism [22], designing polymerase chain reaction (PCR) primer multiplexes in the forensic laboratory [23], amplification and quantitation of human DNA using TPOX locus primers using real-time PCR in the forensic, biochemistry, or molecular biology laboratory [24], and introducing microfluidic gel electrophoresis in the undergraduate laboratory applied to food analysis [25]. Other related papers include an article on DNA profiling from convicted offenders for the Combined DNA Index System (CODIS) [26] and an NSF-funded collaboration between Chemistry and Biology Departments to introduce spectroscopy, electrophoresis, and molecular biology in five different courses [27]. A similar search in Chemical Educator yields a few suitable laboratory experiments adaptable for forensic biology but intended for the biochemistry laboratory including, DNA amplification by PCR and analysis by gel electrophoresis [28] and the application of commercial and web-based software programs for DNA sequence analysis [29]. This journal is also a wealthy source of adaptable laboratory experiments including those designed to have students differentiate bacterial species [30], use the World Wide Web to analyze an Alu insertion polymorphism [31], evaluate a 3STR profile [32], employ PCR in diagnostics [33], test for genetically modified organisms in foodstuffs [34, 35], use real-time PCR to detect lactose resistance [36], perform sex determination using PCR [37], perform ABO blood typing [38], type the HUMTH01 microsatellite [39], and evaluate a SNP in lactose persistence [40], to name a few. In addition to the laboratory experiments described in the aforementioned papers, there are two laboratory manuals Forensic DNA Analysis: A Laboratory Manual published in 2008 [41] and Forensic DNA Biology: A Laboratory Manual published in 2012 [11] that focus on human DNA typing marketed for such courses. Accompanying PowerPoint presentations and an Instructor manual for Forensic DNA Biology: A Laboratory Manual are available from the publisher.

Laboratory Experiments Implemented in this Forensic Biology Course The emphasis of the lab component of the course is to prepare students to work with biological evidence, perform standard assays and DNA typing, and to interpret the results. The associated laboratory sessions are designed to simulate crime scene evidence collection, presumptive testing, and DNA typing of an evidential sample analyzed in order and by the methods used in the crime laboratory setting. The curriculum places a special emphasis on human DNA typing in the forensic lab and the evaluation of imperfect scenarios. In the early years of this curriculum, the laboratory manual for this course was prepared in-house using peer-

Curriculum and Course Materials

reviewed papers from the literature, manuals from commercial kits, and the author’s own creativity. At the time, a commercially available lab manual was unavailable; however, two lab manuals are now available [11, 41], including one by the author. Additionally, there are multiple excellent sources of tools, techniques, protocols, and resources that instructors can access and develop into laboratory experiments for undergraduate students including The Biology Project at The University of Arizona (http://www.biology.arizona.edu/, accessed May 26, 2013), commercial user’s manuals, governmental sources including the National Forensic Science Technology Center’s courses in DNA Analyst Training, Crime Scene Investigation, and Medicolegal Death Investigator training (www.nfstc.org/pdi/), and professional staff at local police departments and state crime laboratories. The laboratory sections used in this curriculum were kept small and ranged from seven to eleven students due to the lab equipment limitations. Sections that exceeded an enrollment of 12 students were split into two lab sections. An overview of the experiments for the 15 laboratory sessions as adopted from Elkins [11] is shown in Table II. The 15-week arrangement (Tables I and II) presented may be optimistic for some instructors and institutions depending upon space, time, and equipment constraints but is a sample schedule we have employed in the past in our courses. I also include a hair and animal fiber microscopy experiment that is not in the lab manual. The sample schedule is designed to complement the order of material presented in lecture. A two semester course would allow for more laboratory experiments and more flexibility with the schedule. An Instructor manual and PowerPoint slides are available to accompany the laboratory manual. A detailed review of the lab experiments is discussed in Table II and overall focuses on simulating the human DNA typing and analysis process as performed by crime laboratories. A few logistical and practical notes follow. As alternative to buccal cells, DNA may be extracted from human hair roots, blood, fingernails, fingerprints, and saliva. Because buccal cells yield a large quantity of DNA, even novices almost always get some material using this method. In addition, non-human DNA can be substituted as a source if needed. The Chelex and PCIA methods are chosen because they are inexpensive and the PCIA method yields the most DNA; however, there are drawbacks: both methods require long incubations and extend across lab periods or weeks. The PCIA method requires a chemical safety hood and the use of gloves as the chemicals can cause detrimental health effects and chemical burns. Commercial DNA extraction kits (e.g. PrepFiler, DNA IQ, and QiaAMP, to name a few) such as used by Frost and Peart [42] are available that minimize the use of organics and do not require a long incubation step; however, their cost can be prohibitive. Some instructors may choose to include only one quantitation method lab due to time constraints. Some labs may be restricted to gel or UV–vis quantitation due to

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the lack of real-time PCR equipment. This instructor prefers to expose the students to multiple methods of quantitation and typically does multiple quantitations as some methods (e.g. UV–vis spectroscopy) give a higher DNA “yield” than real-time PCR with human specific primers. The specificity of the real-time PCR method as compared to the total DNA yield observed with the spectroscopy is a good discussion point. This PCR primer design lab and the subsequent one in which the instructor actually purchases the designed primers so that the students may evaluate their work with real-time PCR are important to the students experiencing science in its truest trial and failure form. The first primers they design often have design flaws based upon the criteria given to them. The creation of multiplexes pushes the students to iteratively work to improve their primer set to better work with another primer set designed by one of their classmates. Students enjoy testing the primers they designed in the lab, and for some, this is their favorite experiment as they have the most ownership in this experiment. Due to the cost of the commercial STR DNA typing kits, this experiment is unfeasible for all but the wealthiest institutions if the kit is not donated. Modifications of this experiment include using an older STR multiplex kit (e.g. Gamma STR by Promega) that creates products than can be evaluated using PAGE, agarose gel electrophoresis using standard molecular biology lab equipment, or using Promega published or instructor designed multiplex primers purchased separately with standard PCR reagents. Likewise, if the focus of the course is more generally on DNA, animal, plant, fungal or bacterial sources may be used with published primers specific to that species [19] or instructor or student-designed primers [23]. Analysis of non-human DNA results must be performed with population databases/tables for that species. Other alternatives to the experiments I have used in my classes include analyzing data for DNA mixtures, X- or Y-chromosome analysis, plant (e.g. cannabis) DNA typing and analysis, or RNA extraction and tissue differentiation [11]. An in silico DNA cloning laboratory is useful to expose students to principles of modern DNA manipulation as covered in some of the introductory forensic textbooks without the use of expensive reagents [44] and can be used to review DNA biology earlier in the course.

Safety In this lab, PCIA reagents for the organic extractions are handled with gloves in the hood. Agarose slab gels are run to avoid handling of polyacrylamide and to facilitate the preparation and electrophoresis of the gels during one laboratory session. SYBR green used to stain the gels is handled as mutagen since it is a DNA intercalating agent but no hazard data is available. All biohazard materials

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Biochemistry and Molecular Biology Education

Laboratory experiments/sessions taught in CHEM 3710 (Criminalistics II)

TABLE II

Chapter xxi, xxv, 1

Week

Laboratory experiment

1

Lab safety, contamination issues: standard laboratory practices, and pipetting.

Lab objectives

Concepts/techniques taught

Lab safety principles and best practices.

Cleaning techniques and standards.

Avoiding contamination.

Evaluating pipette accuracy and precision using a balance.

Introduction to the use of calibrated variable volume micropipettes. Handout

2, 3

2

3

Hair microscopy.

Sampling biological evidence for DNA extraction; Serology.

Identifying cortex, cuticle, and medulla.

Preparing slides for microscopy.

Differentiating hairs from different species/types.

Viewing prepared slides of human/animal hairs.

Practicing proper techniques for collecting and handling biological evidence.

Identification, collection, and packaging techniques using simulated evidence.

Performance of presumptive tests Chemical and immunological for body fluids. methods for presumptive testing of biological fluids. 4

4

DNA Extraction (Week 1).

4

5

DNA Extraction (Week 2).

DNA extraction using Chelex, PCIA, and commercial kit methods.

DNA extraction from buccal cells. DNA extraction from hair root, blood, and saliva. Use of vortex, centrifuge and heating block. Methodological advantages and disadvantages. Safety considerations.

5,6

6

Determination of quality and quantity of DNA.

Use of agarose gel electrophoresis to determine DNA quality and quantity.

Gel preparation and running.

Sample and ladder preparation and loading.

8

7

Real-time polymerase chain reaction (PCR).

Determination of DNA sample concentration using UV–Vis spectrophotometer.

Sample preparation for use in UV–Vis.

Successfully interpret results.

Use of UV–Vis Spectrometer.

Determination of DNA concentration using real-time PCR.

Programming real-time PCR instrument.

Quantitation of DNA

Preparation of sample PCR reaction mixes. Preparation of positive and negative controls.

9

22

8

Design of PCR multiplexes for STR DNA sites of interest.

Use of public databases to obtain DNA locus of interest.

Curriculum and Course Materials

(Continued)

TABLE II

Chapter

Week

Laboratory experiment

Lab objectives

Concepts/techniques taught

Multiplex polymerase chain reaction (PCR) Primer design (in silico). 10

9

Testing designed polymerase chain reaction (PCR).

Evaluation of student-designed PCR multiplexes using real-time PCR.

Design of PCR primer using given parameters.

Evaluation of PCR primers using web-based tools to meet criteria. Primers in multiplex reactions.

Program instrument and run PCR reactions to test primers using appropriate controls. Interpret results.

11

10

Multiplex polymerase chain reaction (PCR) amplification of short tandem repeat (STR) Loci using a commercial kit.

Use of commercial kits for DNA typing.

12

11

Capillary electrophoresis of short tandem repeat (STR) polymerase chain reaction (PCR) products from a commercial multiplex kit.

Separation of PCR products using Preparation of PCR reactions commercial multiplex kits. using kit contents using appropriate positive and negative controls.

Dilution of extracted DNA based on quantitation experiment results.

Amplification of diluted extracted DNA using PCR and commercial multiplex kit primers. Program CE and prepare capillary. Dilute DNA for CE and load samples. Run CE and view data using the instrument software. Analyze DNA STR profile 13,15

12

Computing random match probability from DNA profile data using population databases;

Use of population databases to compute the probability of random match and discriminating power of STR multiplex DNA typing results

Using results from previous labs (Weeks 10–11).

Analysis of DNA Sequence Data Using BioEdit.

View and manipulate DNA sequence data.

Determination of minimum allele frequency for the database provided. Calculation of probability of heterozygous and homozygous allele frequency for the STR profile.

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Biochemistry and Molecular Biology Education

(Continued)

TABLE II

Chapter

Week

Laboratory experiment

Lab objectives

Concepts/techniques taught Calculation of inheritance probability of combined genotypes based on multiple loci using the product rule. Determination of the probability of the genotype in a population for the 13 CODIS loci. Use of software to evaluate and compare DNA sequence data.

14

13

Mitochondrial DNA (mtDNA) single nucleotide polymorphism (SNP) detection.

Evaluate base identity in a mtDNA SNP using real-time PCR.

Programming real-time PCR instrument. Preparation of PCR reaction mixes. Preparation of positive and negative control samples.

19

14

Low copy number stochastic results (Week 1).

Evaluate the results of STR DNA typing using picogram quantities of DNA.

Programming real-time PCR instrument. Preparation of PCR reaction mixes including serial dilutions of DNA. Preparation of positive and negative control samples.

18

14,15 Low copy number stochastic results (Week 2),

Interpret DNA typing results in paternity and missing persons cases using statistics.

Human genetics analysis: paternity of missing persons cases and statistics.

Drawing Punnett squares.

Computing number of genotypes for 13 CODIS loci. Using population databases to determine. Likelihood ratio and combined probability of exclusion. Solving reverse parentage questions.

(e.g. buccal cells, sheep’s blood, etc.) are handled with gloves and collected and autoclaved prior to disposal at the end of the laboratory session. Real-time PCR is performed on a BioRad iQ5 or Applied Biosystems 7000 instrument. Capillary electrophoresis (CE) is performed using an Applied Biosystems 310 or 3130 Genetic Analyzer single capillary electrophoresis system. Student DNA typing data is saved in a secure folder and purged after the completion

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of the experiment. Students are not required to perform their own DNA typing for successful participation and completion of the course. Molecular biology courses are relatively expensive to offer due to the large amount of consumables used (e.g. tips, tubes, reagents, gloves) and because DNA typing kits are costly. Costs can be controlled by having students work in pairs, minimizing the use of commercial kits (e.g. for DNA

Curriculum and Course Materials

extraction and quantitation), accepting donations of equipment and expired kits and reagents (e.g. for DNA extraction, quantitation, and multiplex PCR amplification of STR loci). The computational labs including multiplex PCR primer design, DNA cloning, and analysis of DNA sequence data using BioEdit also minimize costs. They employ computers and free software which are usually accessible on-campus in departmental or campus computer labs or student computers equipped with internet access. Other labs including pipetting, sampling biological evidence, and UV–Vis spectroscopy do not require expensive reagents and utilize equipment typically available in biochemistry and molecular biology laboratories. The majority of the cost is due to PCR and capillary electrophoresis reagents and consumables (e.g. tips, tubes, concentrating devices). Facilities that lack a real-time PCR instrument can perform the experiments using a thermocycler and evaluate the amplified DNA using agarose or polyacrylamide gel electrophoresis. Facilities that lack a capillary electrophoresis or slab DNA sequence instrument can employ older silver stain multiplex kits or homegrown multiplexes that utilize gels for separation instead of a fluorescent dye chemistry kit. Alternatively, if the lab lacks the capabilities to any of the above, sample training data is available on the NIST website (http://www.cstl.nist.gov/biotech/strbase/ training/NIST-MixtureWebcast-Apr2013.htm) including single profiles and DNA mixtures.

Evaluation and Testing Evaluation of student performance in CHEM 3710 was based on four examinations, comprising three exams (administered approximately monthly), a 2-hour final examination, a review research paper and presentation based on a research article from the primary literature, weekly laboratory reports, and weekly quizzes on the previous week’s experiment. The examinations were 20% multiple choice (20 questions) and 80% short answer/essay (7– 10 questions). The research papers were based upon case studies in the primary literature selected from various journals [45–65] including those shown in Table III and approved by the instructor in advance. The students selected a wide range of topics as reflected in the table. The review of the case study paper was due one week prior to the commencement of the students’ presentations of their case in PowerPoint. Laboratory reports are written in a concise format suggested by local crime labs with conservative language suitable for forensic cases.

Outcomes This course curriculum enhances the Bachelor of Science in Chemistry and Forensic Chemistry by exposing students to modern, standardized DNA typing methodologies. Moreover, it provides content required by FEPAC for accreditation purposes, and provides rigorous coverage of the theoretical

Elkins

underpinnings of current crime laboratory practices in the use of molecular biology and biochemistry to identify individuals. The course allows programs to attract high quality students interested in forensic biology and forensic chemistry by offering a current, FEPAC-accredited curriculum, which can be instrumental to the graduates’ career prospects. The Criminalistics program at Metro State is responsible for 32.4% of the chemistry graduates (Bachelor of Arts included) and 55% of the Bachelor of Sciences chemistry majors at the institution. In May 2013 at Towson University, approximately one-quarter of the Bachelor of Science graduates from the Chemistry Department graduated with a degree in Forensic Chemistry (12 Forensic Chemistry graduates and 35 Chemistry graduates, respectively). The Chemistry and Biology Departments anticipate that having our students perform various DNA analyses in the lab based on their lecture experience will increase their motivation to continue their studies in the field. In addition to the course, both departments have developed undergraduate research opportunities working with biological material and DNA. Students are also encouraged to extend beyond their formal coursework training and to attend American Academy of Forensic Sciences (AAFS) National Meetings. Undergraduate students who have taken forensic DNA biology have subsequently become engaged in undergraduate DNA research and have presented posters at the AAFS National meetings.

The Future This article has sought to lay out represents one methodology, “a way,” to collect, synthesize, and instruct what until now has been a largely disparate field of pedagogy. Divided into four broad units, this curriculum is based on the principal of progressive complexity—beginning with basic, rudimentary conceptual information (including theoretical background information) in Unit 1 and progressing to more complex, advanced techniques in Unit 4. In addition, the course seeks to build upon knowledge from more generalized DNA biology courses, but also integrate legal, ethical, and analytical concepts unique to the forensic sciences. In short, this article seeks to provide instructors in FEPACaccredited programs (as well as programs seeking FEPACaccreditation) with a one-stop-shop for teaching forensic DNA biology. The lecture topics, laboratory experiments, as well as the course synchronization matrix can serve as a convenient road map for instructors, and is imminently tailorable and scalable based upon instructor preferences, resource constraints, and time. Finally, this curriculum helps prepare students with critical thinking skills essential for obtaining and retaining professional positions as forensic DNA biologists in a crime lab. By marrying theoretical and contextual knowledge with practical, hands-on training in the laboratory, this methodology will hopefully help train a future cadre of professional forensic DNA biologists

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Biochemistry and Molecular Biology Education

TABLE III

Selected forensic biology journal articles [45–65] used in the forensic biology course

Journal Title Applied and Environmental Microbiology

Titles of Selected Related Forensic Biology Papers [45–65] A field investigation of Bacillus anthracis contamination of U.S. Department of Agriculture and other Washington, D.C. buildings during the anthrax attack of October 2001. Forensic application of microbiological culture analysis to identify mail intentionally contaminated with Bacillus anthracis Spores.

Canadian Society of Forensic Science Journal

Simulated arson experiment and its effect on the recovery of DNA.

Clinica Chimica Acta

De novo deletion at D13S317 locus: A case of paternal–child allele mismatch identified by microsatellite typing. Single and double incompatibility at vWA and D8S1179/D21S11 loci between mother and child: Implications in kinship analysis.

Croatian Medical Journal

World Trade Center Human Identification Project: Experiences with individual body identification cases.

Forensic Science International

The last Viking King: A royal maternity case solved by ancient DNA analysis. Forensic study of stains of blood and saliva in a chimpanzee bite case.

Forensic Science International: Genetics

Two fathers for the same child: A deficient paternity case of false inclusion with autosomal STRs.

International Journal of Legal Medicine

Pitfalls in the analysis of mitochondrial DNA from ancient specimens and the consequences for forensic DNA analysis: the historical case of the putative heart of Louis XVII. Unraveling the mystery of Nanga Parbat. One person with two DNA profiles: a(nother) case of mosaicism or chimerism.

Journal of Clinical Forensic Medicine

Ninhydrin-dyed latent fingerprints as a DNA source in a murder case.

Journal of Forensic Sciences

An illicit love affair during the Third Reich: Who is my grandfather? Integrated DNA and fingerprint analyses in the identification of 60-year-old mummified human remains discovered in an Alaskan glacier.

Molecular Ecology

Patterns of nuclear DNA degeneration over time- a case study in historic teeth samples.

Nature

DNA fingerprints from fingerprints. Jefferson fathered slave’s last child.

Nature Genetics

Mitochondrial DNA sequence heteroplasmy in the Grand Duke of Russia Georgij Romanov establishes the authenticity of the remains of Tsar Nicholas II.

PLoS One

Mystery solved: The identification of the two missing Romanov children using DNA analysis.

The American Journal of Forensic Medicine and Pathology

Identification of exhumed remains of fire tragedy victims using conventional methods and autosomal/Y-chromosomal short tandem repeat DNA profiling.

26

Curriculum and Course Materials

unconstrained by past technological and training limitations, and ultimately improve the quality of forensic crime lab analysis.

Acknowledgements The course at Metro State was made possible by the interactions and input received from Chris Tindall retired from Metro State, Susan Berdine of the Denver Police Department, Kathleen Lobato of the Colorado Bureau of Investigation, and from discussions with practicing forensic analysts at the Front Range Forensic Chemist Meetings in Colorado and Wyoming. Technical representatives from Promega, Applied Biosystems, Beckman Coulter were also helpful in answering questions about their products. Interactions with Amie Ingold and Andrew Carmel were appreciated in regards to improving the courses.

References [1] Butler, J. M. (2005) Forensic DNA Typing: Biology, Technology and Genetics of STR Markers, 2nd ed., Elsevier Academic Press, Burlington, MA.

[17] Jackson, D. D., Abbey, C. S., and Nugent, D. (2006) DNA profiling of the D1S80 Locus: A forensic analysis for the undergraduate biochemistry laboratory, J. Chem. Educ. 83, 774–776. [18] Millard, J. T. and Pilon, A. M. (2003) Identification of forensic samples via mitochondrial DNA in the undergraduate biochemistry laboratory, J. Chem. Educ. 80, 444. [19] Carson, T. M., Bradley, S. Q., Fekete, B. L., Millard, J. T., and LaRiviere, F. J. (2009) Forensic analysis of canine DNA samples in the undergraduate biochemistry laboratory, J. Chem. Educ. 86, 376–378. [20] Taylor, A. and Sajan, S. J. (2005) Testing for genetically modified foods using PCR, J. Chem. Educ. 82, 597–598. [21] Lubin, I. M., Wilson, B., and Grant, K. B. (2003) Allele-specific polymerase chain reaction-based genotyping of a normal variation in human color vision, J. Chem. Educ. 80, 1289–1291. [22] Childs-Disney, J. L., Kauffmann, A. D., Poplawski, S. G., Lysiak, D. R., Stewart, R. J., Arcadi, J. K., and Dinan, F. J. (2010) A metabolic murder mystery: A case-based experiment for the undergraduate biochemistry laboratory, J. Chem. Educ. 87, 1110–1112. [23] Elkins, K. M. (2011) Designing PCR primer multiplexes in the forensic laboratory, J. Chem. Educ. 88, 1422–1427. [24] Elkins, K. M. and Kadunc, R. E. (2012) An undergraduate laboratory experiment for upper-level forensic science courses: The use of TPOX single locus primers to amplify human DNA by real-time PCR with SYBR green detection J. Chem. Educ. 89, 784–790.

[2] Butler, J. M. (2010) Fundamentals of Forensic DNA Typing, Elsevier Academic Press, Burlington, MA.

[25] Chao, T.-C., Bhattacharya, S., and Ros, A. (2012) Microfluidic gel electrophoresis in the undergraduate laboratory applied to food analysis, J. Chem. Educ. 89, 125–129.

[3] Butler, J. M. (2001) Forensic DNA Typing: Biology & Technology Behind STR Markers, Academic Press, San Diego, CA.

[26] Millard, J. T. (2011) DNA profiling of convicted offender samples for the combined DNA index system, J. Chem. Educ. 88, 1385–1388.

[4] Butler, J. M. (2011) Advanced Topics in Forensic DNA Typing: Methodology, Elsevier Academic Press, San Diego, CA.

[27] Bevilacqua, V. L. H., Powers, J. L., Vogelien, D. L., Rascati, R. J., Hall, M., Diehl, K., Tran, C., Jain, S. S., and Chabayta, R. (2002) Collaboration between chemistry and biology to introduce spectroscopy, electrophoresis and molecular biology as tools for biochemistry, R. J. Chem. Educ. 79, 1311.

[5] Deedrick, D.W. and Koch, S. L. (2004) Microscopy of hair. Part 1: A practical guide and manual for human hairs, Forensic Sci. Commun., 6 (1). Available online at http://www.fbi.gov/about-us/lab/forensic-sciencecommunications/fsc/jan2004/research/2004_01_research01b.htm (accessed May 31, 2013). [6] Deedrick, D. W. and Koch, S. L. (2004) Microscopy of hair. Part II: A practical guide and manual for animal hairs, Forensic Sci. Commun., 6 (3). Available online at http://www.fbi.gov/about-us/lab/forensic-sciencecommunications/fsc/july2004/research/2004_03_research02.htm (accessed May 31, 2013). [7] Virkler, K. and Lednev, I. K. (2009) Analysis of body fluids for forensic purposes: From laboratory testing to non-destructive rapid confirmatory identification at a crime scene, Forensic Sci. Int. 188, 1–17. [8] Li, R. (2008) Forensic Biology, CRC Press/Taylor and Francis Group, Boca Raton, FL. [9] Budowle, B., Schutzer, S. E., Breeze, R. G., Keim, P. S., and Morse, S. A., Eds. (2011) Microbial Forensics, 2nd ed., Elsevier Academic Press, Burlington, MA. [10] Gennard, D. (2012) Forensic Entomology: An Introduction, 2nd ed., Wiley-Blackwell, Chichester, West Sussex. [11] Klepinger, L. L. (2006) Fundamentals of Forensic Anthropology, 2nd ed., Wiley, Hoboken, NJ. [12] Elkins, K. M. (2012) Forensic DNA Biology: A Laboratory Manual, Elsevier Academic Press, San Diego, CA. [13] Hartl, D. L. (2000) A Primer of Population Genetics, 3rd ed., Sinauer Associates, Inc., Sunderland, MA. [14] Fung, W. K. and Hu, Y.-Q. (2008) Statistical DNA Forensics: Theory, Methods and Computation, Wiley, Chichester, West Sussex. [15] Goodwin, W., Linacre, A., and Hadi, S. (2011) An Introduction to Forensic Genetics, 2nd ed., Wiley, Chichester, West Sussex. [16] Ruitberg, C. M., Reeder, D. J., and Butler, J. M. (2001) STRBase: A short tandem repeat DNA database for the human identity testing community, Nucleic Acids Res. 29, 320–322.

Elkins

[28] Ooi, B. G. (2004) DNA amplification by PCR and analysis by gel electrophoresis—An undergraduate biochemistry experiment, Chem. Educ. 9, 97–101. [29] Ooi, B. G. (2005) Application of commercial and web-based software programs for DNA sequence analysis in undergraduate biochemistry laboratory, Chem. Educ. 10, 186–189. [30] Suwanjinda, D., Eames, C., and Panbangred, W. (2007) Screening of lactic acid bacteria for bacteriocins by microbiological and PCR methods, Biochem. Mol. Biol. Educ. 35, 364–369. [31] Kass, D. H. and LaRoe, R. (2007) Web-based analysis for student-generated complex genetic profiles, Biochem. Mol. Biol. Educ. 35, 404–409. [32] McNamara-Schroeder, K., Olonan, C., Chu, S., Montoya, M. C., Alviri, M., Ginty, S., and Love, J. J. (2006) DNA fingerprint analysis of three short tandem repeat (STR) loci for biochemistry and forensic science laboratory courses, Biochem. Mol. Biol. Educ. 34, 378–383. [33] Claros, M. G. and Quesada, A. R. (2000) PCR as a specific, sensitive and simple method suitable for diagnostics, Biochem. Mol. Biol. Educ. 28, 223–226. [34] Thion, L., Vossen, C., Couderc, B., Erard, M., and Clemenc¸on, B. (2002) Detection of genetically modified organisms in food by DNA extraction and PCR amplification, Biochem. Mol. Biol. Educ. 30, 51–55. [35] Brinegar, C. and Levee, D. (2004) A simple method for detecting genetically modified maize in common food products, Biochem. Mol. Biol. Educ. 32, 35–38. [36] Weinlander, K. M., Hall, D. J., and De Stasio, E. A. (2010) RFLP analysis and allelic discrimination with real-time PCR using the human lactase persistence trait, Biochem. Mol. Biol. Educ. 38, 167–171. [37] Kima, P. E. and Rasche, M. E. (2004) Sex determination using PCR, Biochem. Mol. Biol. Educ. 32, 115–119.

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Biochemistry and Molecular Biology Education [38] Martin, M. P. and Detzel, S. M. (2008) A laboratory exercise to determine human ABO blood type by noninvasive methods, Biochem. Mol. Biol. Educ. 36, 139–146. [39] Lajoie-Mazenc, I. and Couderc, B. (2001) Analysis of the tetranucleotide polymorphic HUMTH01 microsatellite, Biochem. Mol. Biol. Educ. 29, 71–75. [40] Schultheis, P. J. and Bowling, B. V. (2011) Analysis of a SNP linked to lactase persistence: An exercise for teaching molecular biology techniques to undergraduates, Biochem. Mol. Biol. Educ. 39, 133–140. [41] McClintock, J. T. (2008) Forensic DNA Analysis: A Laboratory Manual, 1st ed., CRC Press/Taylor and Francis Group, Boca Raton, FL. [42] Frost, L. D. and Peart, S. T. (2003) DNA isolation from a dried blood sample, PCR amplification, and population analysis: Making the most of commercially available kits, Biochem. Mol. Biol. Educ. 31, 418–421. [43] Budowle, B., Moretti, T. R., Baumstark, A. L., Defenbaugh, D. A., and Keys, K. M. (1999) Population data on the thirteen CODIS core short tandem repeat loci in African Americans, US Caucasians, Hispanics, Bahamians, Jamaicans, and Trinidadians, J. Forensic Sci. 44, 1277– 1286. [44] Elkins, K. M. (2011) An in silico DNA cloning experiment for the biochemistry laboratory, Biochem. Mol. Biol. Educ. 39, 211–215. [45] Higgins, J. A., Cooper, M., Schroeder-Tucker, L., Black, S., Miller, D., Karns, J. S., Manthey, E., Breeze, R., and Perdue, M. L. (2003) A field investigation of Bacillus anthracis contamination of U.S. Department of Agriculture and other Washington, D.C. buildings during the anthrax attack of October 2001, Appl. Environ. Microbiol. 69, 593–599. [46] Beecher, D. J. (2006) Forensic application of microbiological culture analysis to identify mail intentionally contaminated with Bacillus anthracis spores, Appl. Environ. Microbiol. 72, 5304–5310. [47] Abrams, S., Reusse, A., Ward, A., and Lacapra, J. (2008) A simulated arson experiment and its effect on the recovery of DNA, Can. Soc. Forensic Sci. 41, 53–60. [48] Narkuti, V., Oraganti, N. M., Vellanki, R. N., and Mangamoon, L. N. (2009) De novo deletion at D13S317 locus: A case of paternal–child allele mismatch identified by microsatellite typing, Clin. Chim. Acta 403, 264–265. [49] Narkuti, V., Vellanki, R. N., Anubrolu, N., Doddapaneni, K. K., Kaza, P. C. G., and Mangamoori, L. N. (2008) Single and double incompatibility at vWA and D8S1179/D21S11 loci between mother and child: Implications in kinship analysis, Clin. Chim. Acta 395, 162–165. [50] Budimlija, Z. M., Prinz, M. K., Zelson-Mundorff, A., Wiersema, J., Bartelink, E., MacKinnon, G., Nazzaruolo, B. L., Estacio, S. M., Hennessey, M. J., and Shaler, R. C. (2003) World trade center human identification project: Experiences with individual body identification cases, Croatian Med. J. 44, 259–263. [51] Dissing, J., Binladen, J., Hansen, A., Sejrsen, B., Willerslev, E., and Lynnerup, N. (2007) The last Viking King: A royal maternity case solved by ancient DNA analysis, Forensic Sci. Int. 166, 21–27. [52] Tsutsumi, H. and Katsumata, Y. (1993) Forensic study of stains of blood and saliva in a chimpanzee bite case, Forensic Sci. Int. 61, 101–110.

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lez-Andrade, F., Sa nchez, D., Penacino, G., and Jaretta, B. M. [53] Gonza (2009) Two fathers for the same child: A deficient paternity case of false inclusion with autosomal STRs, Forensic Sci. Int. Genet. 3, 138–140. [54] Jehaes, E., Toprak, K., Vanderheyden, N., Pfeiffer, H., Cassiman, J.-J., Brinkmann, B., and Decorte, R. (2001) Pitfalls in the analysis of mitochondrial DNA from ancient specimens and the consequences for forensic DNA analysis: The historical case of the putative heart of Louis XVII, Int. J. Legal Med. 115, 135–141. [55] Parson, W., Brandstatter, A., Niederstatter, H., Grubwieser, P., and Scheithauer, R. (2007) Unravelling the mystery of Nanga Parbat, Int. J. Legal Med. 121, 309–310. [56] Castella, V., del Mar Lesta, M., and Mangin, P. (2009) One person with two DNA profiles: a(nother) case of mosaicism or chimerism, Int. J. Legal Med. 123, 427–430. [57] Schulz, M. M., Wehner, H.-D., Reichert, W., and Graw, M. (2004) Ninhydrin-dyed latent fingerprints as a DNA source in a murder case, J. Clin. Forensic Med., 11, 202–204. [58] Milde-Kellers, A., Krawczak, M., Augustin, C., Boomgaarden-Brandes, K., Simeoni, E., Kaatsch, H.-J., M€ uhlbauer, B., and Schuchardt, S. (2008) An illicit love affair during the Third Reich: Who is my grandfather? J. Forensic Sci. 53, 377–379. [59] Loreille, O. M., Parr, R. L., McGregor, K. A., Fitzpatrick, C. M., Lyon, C., Yang, D. Y., Speller, C. F., Grimm, M. R., Grimm, M. J., Irwin, J. A., and Robinson, E. M. (2010) Integrated DNA and fingerprint analyses in the identification of 60-year-old mummified human remains discovered in an Alaskan glacier, J. Forensic Sci. 55, 813–818. [60] Wandeler, P., Smith, S., Morin, A., Pettifor, R. A., and Funk, S. M. (2003) Patterns of nuclear DNA degeneration over time: A case study in historic teeth samples, Mol. Ecol. 12, 1087–1093. [61] van Oorschot, R. A. and Jones, M. K. (1997) DNA fingerprints from fingerprints, Nature 387, 767. [62] Foster, E. A., Jobling, M. A., Taylor, P. G., Donnelly, P., de Knijff, P., Mieremet, R., Zerjal, T., and Tyler-Smith, C. (1998) Jefferson fathered slave’s last child, Nature 396, 27–28. [63] Ivanov, P. L., Wadhams, M. J., Roby, R. K., Holland, M. M., Weedn, V.W., and Parsons, T. J. (1996) Mitochondrial DNA sequence heteroplasmy in the Grand Duke of Russia Georgij Romanov establishes the authenticity of the remains of Tsar Nicholas II, Nat. Genet. 12, 417–420. [64] Coble, M. D., Loreille, O. M., Wadhams, M. J., Edson, S. M., Maynard, K., Meyer, C. E., Niederstatter, H., Berger, C., Falsetti, A. B., Gill, P., Parson, W., and Finelli, L. N. (2009) Mystery solved: The identification of the two missing Romanov children using DNA analysis, PLoS One 4, e4838. [65] Calacal, G. C., Delfin, F. C., Tan, M. M. M., Roewer, L., Magtanong, D. L., Lara, M. C., Fortun, Rd., and De Undria, M. C. (2005) Identification of exhumed remains of fire tragedy victims using conventional methods and autosomal/Y-chromosomal short tandem repeat DNA profiling, Am. J. Forensic Med. Pathol. 26, 285–291.

Curriculum and Course Materials