Biotechnology Explorer™ Crime Scene Investigator PCR Basics™ Kit Catalog #166-2600EDU explorer.bio-rad.com Note: Kit contains temperature-sensitive reagents. Open immediately upon arrival and store components at –20°C or at 4°C as indicated. Duplication of any part of this document is permitted for classroom use only. Please visit explorer.bio-rad.com to access our selection of language translations for Biotechnology Explorer kit curricula. For technical service, call your local Bio-Rad office or, in the U.S., call 1-800-424-6723 Crime Scene Investigator PCR Basics™ Kit This curriculum was developed in collaboration with Dr. Linda Strausbaugh, Director of the Center for Applied Genetics and Technology at the University of Connecticut-Storrs. This Crime Scene Investigator PCR Basics kit allows students to conduct state-of-the-art DNA pro- filing techniques and to develop an understanding of how these techniques are performed in real-world forensic science labs. Bio-Rad is grateful for Dr. Strausbaugh’s guidance and mentorship. How can DNA evidence solve crimes? DNA profiling is the use of molecular genetic methods to determine the exact genotype of a DNA sample to distinguish one human being from another. This powerful tool is now routinely used around the world for investigations of crime scenes, missing persons, mass disasters, human rights violations, and paternity testing. Crime scenes often contain biological evidence (such as blood, semen, hairs, saliva, bones, pieces of skin) from which DNA can be extracted. If the DNA profile obtained from evidence discovered at the scene of a crime matches the DNA profile of a suspect, the individual is included as a potentially guilty person; if the two DNA profiles do not match, the individual is excluded from the suspect pool. What kinds of human DNA sequences are used in crime scene investigations? There are ~3 billion bases in the human genetic blueprint – greater than 99.5% do not vary among human beings. However, a small percentage of the human DNA sequence (<0.5%) does differ, and these are the polymorphic ("many forms") sequences used in forensic applications. By universal agreement, the DNA sequences used for forensic typing are "anonymous"; that is, they are derived from regions of our chromosomes (also called loci) that do not control any known traits and have no known functions. The DNA sequences used in forensic DNA profiling are non-coding regions that contain segments of short tandem repeats or STRs. STRs are very short DNA sequences that are repeated in direct head-to-tail fashion. The example below (Figure 1) shows a locus known as TH01) actually used in forensic DNA profiling; the DNA sequence at this locus contains four repeats of [TCAT]. For the TH01 STR locus, there are many alternate polymorphic forms (alleles) that differ from each other by the number of [TCAT] repeats present in the sequence. Although more than 20 different alleles of TH01 have been discovered in people worldwide, each of us still has only two of these, one inherited from our mother and one inherited from our father. Suspect A’s DNA type for the TH01 locus is (5–3) Suspect B’s DNA type for TH01 locus is (6–10) C C C A A A 5* C C C A A A 6* C C C A A A 3* C C C A A A 10* * Number of [TCAT] repeats Fig. 1. Two sample genotypes at the TH01 locus. How are STR alleles detected? The key to DNA profiling is amplification of the copies present in the small amounts of evidentiary DNA by polymerase chain reaction (PCR). Using primers specific to the DNA sequences on either side of the STR, billions of copies of each of the two original TH01 alleles in any one person's DNA type are synthesized in the reaction. These copies contain the same number of STRs present in the original DNA copies and can be separated by size using agarose gel electrophoresis. By comparison with size standards that correspond to the known sizes of TH01 alleles, the sizes of the amplified copies can be determined. This kit allows students to simulate genotyping at a locus as commonly used in forensic typing. In real crime scene applications, DNA profiling is performed at a number of different loci to improve the power of discrimination of the testing. In simple terms, the power of discrimination is the ability of the typing to discriminate between different individuals. As an example, one locus may be able to tell the difference between one out of 1,000 people, whereas two loci can discriminate between one out of 10,000 people. The larger the number of loci typed, the more powerful the ability to discriminate. Following the DNA profiling at the simulated BXP007 locus in the Crime Scene Investigator PCR Basics kit, the teacher has the option of an additional lesson that simulates the use of the 13 core CODIS (Combined DNA Index System) loci used in actual casework. This simple exercise demonstrates the concept of increasing power of discrimination with increasing numbers of loci typed, and illustrates how even siblings can be identified by DNA profiling. Each student team will generate their own set of genotypes, collect and record data on a worksheet, and perform simple statistical calculations. Teaching Strategy – Guided Inquiry-Based Investigation The intent of this curriculum is to guide students through the thought process involved in a laboratory-based scientific investigation. Students will learn about PCR, gel electrophoresis, genotyping, and genotype matching while asking the question "How can a tiny amount of genetic material (DNA) be used to identify one person out of a billion?" In the context of a crime scene case study scenario, this activity could raise a host of questions such as "Why is this important?" The students' focus is not so much on the answer or result, but on how the result was obtained and substantiated by careful observation and analysis of their data. Students who engage in Biotechnology Explorer activities develop a positive sense of their ability to employ the scientific method to solve problems. Thought provoking questions embedded in the student manual are designed to maximize students' involvement in the laboratory. Student involvement in this process results in an increased understanding of the value of approaching a scientific challenge in an organized and logical fashion. This manual can be downloaded from the Biotechnology Explorer website. Visit us on the web at explorer.bio-rad.com, or call us in the US at 1-800-4BIORAD (1-800-424-6723). We continually strive to evolve and improve our curricula and products. We welcome your suggestions and ideas! Biotechnology Explorer Program Life Science Group Bio-Rad Laboratories Hercules, CA 94547 explorer.bio-rad.com Create context. Reinforce learning. Stay current. New scientific discoveries and technologies create more content for you to teach, but not more time. Biotechnology Explorer kits help you teach more effectively by integrating multiple core content subjects into a single lab. Connect concepts with techniques and put • Use of PCR and gel electrophoresis them into context with for DNA profiling • Use of experimental controls real-world scenarios. • Interpretation of experimental results • Use of forensic evidence in the courts Environmental Scientific and Health Inquiry Science • Population genetics • Genetic screening • Genetic databases Crime Scene • Role, place, limits of science and technology Investigator Chemistry • Privacy of information issues PCR Basics of Life Kit • DNA replication and the polymerase chain reaction (PCR) • Chemical properties of biological • Eukaryotic cell structure Cell and and organization molecules • Tissue types and biological Molecular Genetics • DNA structure and function sampling Biology • Chemistry of electrophoresis • Genetic diversity and individual identification • Mendelian genetics • Allele frequencies in Evolution • Polymorphic loci and multiple alleles populations • Genetics of noncoding DNA • Statistics, probabilities, and • Short tandem repeats (STRs) the power of discrimination • Genetic identity • Structure and function of the human genome Table of Contents Page Kit Summary ....................................................................................................................1 Kit Inventory Checklist ......................................................................................................2 Curriculum Fit ..................................................................................................................4 Background for Instructors................................................................................................6 Instructor's Advance Preparation ....................................................................................13 Lesson 1: Set Up PCR Reactions ............................................................................15 Lesson 2: Electrophoresis of PCR Products ............................................................17 Lesson 3: Drying Gels and Analysis of Results ........................................................21 Tips and Frequently Asked Questions ............................................................................23 Quick Guide ..................................................................................................................27 Student Manual ..............................................................................................................30 Lesson 1: PCR Amplification....................................................................................35 Lesson 2: Electrophoresis