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DNA: The Indispensable Forensic Science Tool Chapter 9 Introduction 1985-Sir Alex Jeffreys recognized that portions of DNA structure of certain genes are unique to each individual. The process of identifying these markers is known as DNA fingerpringting, DNA typing, or DNA profiling Forensic Scientists now had a way of linking biological samples like blood, semen, hair, and tissue to a single individual US courts have accepted the reliability of DNA evidence DNA = Deoxyribonucleic Acid DNA is a polymer Polymers are very large molecules put together by linking units called monomers The monomers of DNA are called nucleotides. Nucleotides DNA nucleotides consist of : A sugar molecule that forms the backbone A phosphate group which links the sugars A nitrogen containing base (adenine, cystine, guanine, thymine) Nucleotides are linked together to form a double helix What is DNA? Complementary Base Pairing Adenine always pairs with Thymine Guanine always pairs with Cytosine Bases are joined together by hydrogen bonding. How DNA works DNA directs the production of proteins which play a basic role in the structure and function of living organisms Proteins are built by linking amino acids The are 20 amino acids that make up proteins The sequence of amino acids determines the shape and function of the protein How DNA works Each amino acid is coded by a sequence of 3 bases Replication of DNA Replication – synthesis of new DNA from existing DNA The double helix is unwound and a copy is made. DNA polymerase – Assemble new DNA strands in the proper base sequence Correct mismatched base pairs Polymerase Chain Reaction (PCR) A technique for replicating a portion of DNA outside of a living cell DNA polymerases are used to make copies of DNA material This is useful to forensic scientists because small samples could be multiplied. DNA Typing with Tandem Repeats Tandem repeat –sequence of base pairs repeated numerous times. ~30 % of human genome is made up of tandem repeats. They act as spacers in between coding regions of the DNA Used by forensic scientists to distinguish individuals. DNA Typing with Tandem Repeats Humans have the same type of repeats but a wide variety in the number of repeats. Restriction fragment length polymorphisms (RFLP’s) – repeat segments cut out of DNA by restriction enzymes. DNA Typing with Tandem Repeats Length differences associated with RFLP’s allow forensic scientists to identify individuals. Electrophoresis is used to separate different sized fragments. Gel Electrophoresis DNA Typing with Tandem Repeats Polymerase Chain Reaction (PCR) PCR replaced RFLP techniques in the mid1990’s The Basics: Short DNA sequences are identified. DNA strands are unwound and replicated yielding two copies of the original sample 28-32 cycles are carried out yielding over a billion copies of the DNA strand. Polymerase Chain Reaction Polymerase Chain Reaction (PCR) Advantages of PCR over RFLP typing: 1. PCR uses much smaller strands of DNA 2. Smaller strands are more stable and less subject to degradation 3. Smaller strands overcome the samplesize problem associated with crime-scene evidence Short Tandem Repeats (STR’s) STR’s – DNA profiling procedure used currently Short Tandem Repeats – regions of DNA molecules containing short segments of 3 to 7 repeating base pairs. STR’s are less susceptible to degradation and can be amplified by PCR Multiplexing: A technique that simultaneously detects more than one DNA marker in a single analysis. Short Tandem Repeats (STR’s) In the US, the forensic science community has standardized 13 STR’s for entry into the CODIS database FBI Core STR Loci Calculating the frequency of occurence of a DNA profile Separation of STR's by capillary electrophoresis Results of electrophoresis on STR's Sex Identification Using STR’s The sex of a DNA contributor can be determined by looking at the amelogenin gene. The gene is 6 bases shorter on the X chromosome than it is on the Y chromosome. Y-STR’s – STR’s found on the Y chromosome; useful when there is a mix of DNA from more than one male. Mitochondrial DNA Mitochondrial DNA (mtDNA) – found in the mitochondria (outside of the cell nucleus) mtDNA is inherited solely from the mother Forensic Scientists use mtDNA to identify subjects when nuclear DNA is not available. Frequencies of sequences in the FBI database are about 1 % inheritance of mtDNA structure of mtDNA The Combined DNA Index System (CODIS) CODIS – software program and database developed by the FBI Contains DNA profiles of convicted offenders, unsolved crime-scene evidence, and profiles of missing people. Collection and Preservation of DNA Evidence STR profiling requires only 125 picograms of DNA One human cell contains ~ 7 pg of DNA Only 18 cells are necessary As a result, DNA has been successfully analyzed from stamps, envelopes, cups, cigarette butts, chewing gum, skin cells, etc. Collection and Preservation of DNA Evidence Biological evidence should be documented by notes, sketches, and photographs first The shape and position of bloodstains should be investigated prior to collection. Latex gloves will protect investigators from infection AND avoid contaminating the evidence. Items like clothing should be collected from suspects and victims even if blood isn’t immediately visible. Collection and Preservation of DNA Evidence Packaging of Evidence: Biological evidence should not be packaged in an airtight container; paper bags, envelopes, or cardboard boxes can be used. Samples should be refrigerated and kept out of direct sunlight Blood stains can be removed from a surface using a cotton swab soaked in distilled water. Swabs must be dried prior to packaging Collection and Preservation of DNA Evidence Obtaining DNA Reference Samples: Reference samples can be obtained by collecting blood or a buccal swab from an individual If the individual is not available: Tootbrush Combs and hairbrushes Razor Dirty laundry Cigarette butts Collection and Preservation of DNA Evidence To minimize contamination: Change gloves before handling each new piece of evidence Collect a substrate control. Use forceps to pick up small items Package items is individual well-ventilated containers. Luminol does not destroy DNA