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Chapter 9 DNA: THE INDISPENSIBLE FORENSIC SCIENCE TOOL FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-1 Introduction • The gene is the fundamental unit of heredity. • Each gene is actually composed of a DNA sequence that specifically code for a protein. • DNA is a polymer made by linking a series of repeating units called nucleotides (monomers). • Each nucleotide is composed of a sugar, a phosphate group, and a nitrogen base. FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-2 The Bases • Four types of bases used in DNA: adenine (A), guanine (G), cytosine (C), and thymine (T). • DNA’s structure is a double-stranded helix as discovered by Rosalind Franklin, Crick and Watson. • Chargaff discovered that A always pairs with T and C always pairs with G – AKA base pairing. FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-3 DNA at Work • DNA codes for proteins ( also polymers), which are formed by amino acids (monomers) in a long chain. • The sequence of amino acids determines the shape and function of the protein. • A codon (three nucleotides) codes for a particular amino acid. G-A-G codes for the amino acid glutamine, while CFORENSIC SCIENCE An Introduction G-T codes for By Richard Saferstein • G-A-G codes for glutamine, C-G-T codes for alanine. PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-4 Nucleotide Errors • If a nucleotide is changed or omitted, a T is substituted for A then G-A-G becomes G-T-G, and a different amino acid is placed in that position in the protein (in this case: glutamine is replaced with valine). • As a result, the protein may not function correctly and this is the basis for many diseases and health issues. FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-5 Intracellular DNA Replication • DNA duplicates itself in the nucleus prior to cell division. • DNA replication begins with the unwinding of the DNA strands of the double helix. • Many enzymes are involved in unwinding the DNA (helicases), and assembling the new DNA strands (polymerases). FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-6 Extracellular DNA Replication • Polymerase chain reaction (PCR) is a technique for replicating a small DNA sample found at a crime scene. • The ability to multiply small bits of DNA means that a single sample is no longer a limitation in analyzing crime scene DNA. FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-7 PCR • PCR uses the enzyme taq-polymerase (taken from bacteria), and Adenine, Thymine, Cytosine and Guanine to copy a segment of DNA. • Very little DNA is needed (0.2 ul) FORENSIC SCIENCE An Introduction By Richard Saferstein • All the ingredients are placed into the thermal-cycler. • Thermal-cycler heats the DNA sequence to unravel the strand. • The A, T, C and G will pair with the sample in the correct order, the copy will be released when the cycle cools PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-8 Recombinant DNA • Recombinant DNA relies on the ability of proteins, known as restriction enzymes to cut through both strands in DNA sequence. • Restriction enzymes can be thought of as highly specialized scissors that cut a DNA molecule when recognizing a specific sequence of bases. • Once a portion of the DNA strand has been cut out with the aid of a restriction enzyme, the next step is to insert the DNA fragment into another DNA strand (usually in bacteria). • The bacteria divide rapidly passing the newly-altered DNA on to all new bacteria. • Method used to produce insulin, penicillin, botox. FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-9 Recombinant DNA Recombinant Cloning: Plasmid insertion of cut DNA sequence. New piece of DNA is incorporated into the bacterial DNA. FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-10 DNA Typing • Portions of the DNA molecule contain sequences of bases that are repeated numerous times, known as tandem repeats. • To a forensic scientist, these tandem repeats offer a means of distinguishing one individual from another through DNA-typing. • Tandem repeats are found throughout our genome between the coding areas of DNA. • What is important to understand is that all humans have the same repeat regions, but there is tremendous variation in the number of repeats and each person has a unique number in each region of their genome. FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-11 DNA Typing FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-12 RFLP • Long repeating DNA sequences are called restriction fragment length polymorphisms (RFLP’s) and form the basis for the first DNAtyping procedure. • The sequence consists of a range of 15-40 basepairs in length and can be repeated 20-1000 times in each region. • The key to understanding DNA typing is that total number of repeats of the specific region is inherited from each parent to form a unique combination for each child. FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-13 RFLP Analysis FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-14 Electrophoresis • A technique analogous to TLC is electrophoresis. – Here, materials are forced to move across a gel-coated plate under the influence of an electrical potential. • In this manner, substances such as DNA can be separated and characterized. FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-15 Agarose Gel Electrophoresis FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-16 DNA Typing • Once the DNA molecules have been cut up by a restriction enzyme, the resulting fragments are sorted out by electrophoresis. • The smaller DNA fragments will move at a faster rate through the electrical field than the larger ones. • The fragments are then transferred to a nylon membrane in a process called Southern blotting. • To visualize the RFLPs, the nylon sheet is treated with radioactive (or bioluminescent) probes containing a base sequence complementary to the RFLPs being identified (a process called hybridization). FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-17 DNA Typing • Next, the nylon sheet is placed against X-ray film and exposed for several days. • When the film is processed, dark bands will appear where radioactive probes stuck to fragments on the nylon sheet. • A typical DNA fragment pattern will show two bands (one RFLP from each chromosome). • When comparing the DNA fragment patterns of two or more specimens, one merely looks for a match between the band sets. • A high degree of discrimination can be achieved by using a number of different probes and combining their frequencies. FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-18 PCR Testing • Polymerase chain reaction is based on the way DNA strands naturally replicate within a cell. • In forensics, PCR offers a distinct advantage in that it can amplify minute quantities of DNA many millions of times. • First, the DNA is heated to separate it. • Second, primers (short strands of DNA used to target specific regions of DNA for replication) are added, which hybridize with the strands. • Third, DNAtaq- polymerase and free nucleotides (A,T,C,G) are added to the mixture to rebuild each of the separated strands. • The heating-cooling cycles are repeated 25 to 30 times. FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-19 PCR Technology FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-20 PCR and RFLP • PCR technology cannot be applied to RFLP DNA typing. • The RFLP strands are too long, often numbering in the thousands of bases. • PCR is best used with DNA strands that are no longer than a couple of hundred bases. FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-21 PCR Advantages • First advantage in moving to shorter DNA strands is that they are more stable and less subject to degradation (breakdown) due to adverse environmental conditions. • Second advantage is long RFLP strands tend to break apart under the adverse conditions (which are all too common at crime scenes). • Third advantage is PCR can amplify minute (tiny) quantities of DNA, producing an unlimited supply of reliable crime scene evidence. FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-22 Short Tandem Repeats • The CURRENT method of DNA typing, short tandem repeat (STR) analysis is the most successful and widely used DNA procedure. • STRs are locations on the chromosome that contain short sequences that repeat themselves inside the DNA code. • They are valuable markers for identification because they are located throughout the human genome. • CODIS requires 13 standard STR sites for submission. FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-23 STR Advantages • STRs normally consist of repeating sequences of 3 to 7 bases in length. The entire strand of an STR is very short, less than 450 bases in length. • This means that STRs are much less susceptible to degradation and may often be recovered from bodies or stains that have been subjected to extreme decomposition. • Due to shortened sequences, STRs are easily amplified by PCR. FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-24 The Power of STR • Hundreds of STRs are found in human genes. • The more STRs that can be matched to a suspect, the more individual the evidence. • Now forensic labs wil perform multiplexing DNA testing, which will identify more correlation between the suspect and the crime. • PCR allows the scientist to simultaneously extract and amplify a combination of different STRs. FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-25 Standardizing STR Testing • Currently, U.S. crime laboratories have standardized on 13 STRs for entry into a national database (CODIS). • The high degree of discrimination and even individualization can be attained by analyzing a combination of STRs (multiplexing) and determining the product of their frequencies. • With STR, as little as 125 picograms of DNA is required for analysis. • This is 100 times less than that normally required for RFLP analysis. FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-26 Mitochondrial DNA • Another type of DNA used for individual characterization is mitochondrial DNA. • Mitochondrial DNA (mDNA) is located outside the cell’s nucleus and is inherited from the mother. • Mitochondria are structures found in all our cells used to provide energy that our bodies need to function. • A single mitochondria contains several loops of DNA. FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-27 Mitochondrial DNA Testing • Mitochondrial DNA typing does not approach STR analysis in its discrimination power and thus is best reserved for samples, such as hair, for which STR analysis may not be possible. • Forensic analysis of mDNA is more rigorous, time consuming, and costly when compared to nuclear DNA analysis. • Also, all individuals of the same maternal lineage will be indistinguishable by mDNA analysis. • Two regions of mDNA have been found to be highly variable and a procedure known as sequencing is used to determine the order of base pairs. FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-28 CODIS • Perhaps the most significant development to arise from DNA typing is the ability to compare DNA types recovered from crime scene evidence to those of convicted sex offenders and other criminals. • CODIS (Combined DNA Index System) is a computer software program developed by the FBI that maintains local, state, and national databases of DNA profiles from convicted offenders, unsolved crime scene evidence, and profiles of missing persons. FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-29 Packaging Biological Evidence • Before the collection of biological evidence begins, it is important that it be photographed and recorded on sketches. • Wearing disposable latex gloves while handling the evidence is required. • Clothing from victim and suspect with blood evidence must be collected. • The packaging of biological evidence in plastic or airtight containers must be avoided because the accumulation of residual moisture could contribute to the growth of DNA-destroying bacteria and fungi. FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-30 Packaging Biological Evidence • Each stained article should be packaged separately in a paper bag or in a well-ventilated box. • Dried blood is best removed from a surface by using a sterile cotton swab lightly moistened with distilled water that is air dried before being placed in a swab box, then a paper or manila envelope. • All biological evidence should be refrigerated or stored in a cool location until delivery to the laboratory. • Standard/reference DNA specimens must also be collected, such as blood or the buccal swab (swabbing the mouth and cheek). FORENSIC SCIENCE An Introduction By Richard Saferstein PRENTICE HALL ©2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 9-31