1. DNA: the indispensable forensic science tool

2. What is DNA Deoxyribonucleic Acid – Genetic Code of Life Codes for production of proteins that determines our traits

3. Where is DNA? Prokaryotic organisms = bacteria – DNA is located as one large circular strand (chromosome) within the cell Eukaryotic Organisms = plants, animals, fungi – Condensed DNA forms chromosomes and is found within the nucleus of the cell Humans have 46 chromosomes DNA wraps around proteins called histones to condense and form chromosomes

4. Structure of DNA Structure is a DOUBLE HELIX or “twisted ladder” – DNA is a large macromolecule (polymer) made of nucleotide monomers Each nucleotide is composed of a deoxyribose (sugar), a phosphate group, and nitrogen base

5. Structure of DNA Sides of the ladder are made of alternating deoxyribose (sugar) and phosphate groups. Nitrogenous Bases—pairs of molecules that form the rungs of the DNA “ladder” – Four types of Bases A (adenine) C (cytosine) G (guanine) T (thymine) – Base pairing rules A (adenine) always bonds/pairs with T (Thymine) C (cytosine) always bonds with G (Guanine)

6. DNA at Work DNA is the genetic code for producing all the different proteins needed by the body – Roles of proteins Enzymes- speed up chemical reaction Cell Transport- movement of materials in and out of the cell Structural- form parts of the body Fight disease ect… Transcription and Translation – Genes are segments of DNA that code for a particular trait Genes are transcribed into mRNA mRNA is then translated by ribosome into the amino acid sequence of proteins

7. DNA Replication DNA Replication- Process in which DNA is copied, process is semiconservative – Occurs before a new somatic (body) can be created Each cell needs a copy of instructions – Occurs before production of gametes or reproductive cells – Sperm – Egg

8. Polymerase Chain Reaction Laboratory process of making copies of DNA – Forensic scientists can make billions of copies of small DNA samples in just a few minutes – Process mimics DNA Replication Steps 1DNA Heated to separate two strands 2Add primer and lower temperature -primer = short strand of complementary DNA that will target and attach to original separated strands 3Add free nucleotides and DNA polymerase -DNA polymerase attaches free nucleotides to remaining original strands to form new “complementary strand result is two molecules of DNA that are identical to original – Each has one original strand and 1 new complementary strand, therefore replication is said to be “semiconservative”

9. Restriction Enzymes Chemical Agents that cut DNA at specific sites Many different Restriction Enzymes exist that cut at specific locations – Example EcoRi – G A A T T C C T T A A G

10. RFLP’s Restriction Fragment Length Polymorphisms Takes advantage of the fact RESTRICTION ENZYMES will cut DNA at specific sites Specific RESTRICTION ENZYMES can be used to cut into DNA at these specific locations Creates different sized fragments that can be separated by gel electrophoresis

11. RFLP Analysis R – Restriction enzymes are used to cut known segments of DNA into F – Fragments that are many different L – Lengths and exhibit P – Polymorphisms, which is the Greek term meaning many sizes. The length of the fragments will vary greatly among individuals

12. dna fingerprinting Uses process of gel electrophoresis – DNA sample that has been cut is place into wells at ends of gel agarose – Gel agarose (jello like substance) used to separate DNA into different sized fragments – Creates pattern (fingerprint)

13. Steps of DNA Fingerprinting 1.Extraction -Cells are isolated and treated with chemicals to release chromosomes from the nucleus -Chromosomes are unwrapped and DNA is unraveled *enzymes destroy histones in which DNA is coiled around 2.DNA is cut with restriction enzymes -different restriction enzymes recognize and cut at different sequences example: EcoRi recognition sequence = GAATTC

14. Steps of DNA Fingerprinting 3. DNA fragments are separated by the process of gel electrophoresis -DNA negatively charged and attracted to positive end -shorter fragments travel farther 4. DNA stains are used to show banding pattern or -probes (often radioactive) that attach to DNA can be used and a film can later be developed to show pattern

15. Sample DNA Fingerprint Analysis

16. DNA Fingerprinting DNA fingerprinting can – match crime scene DNA with a suspect – determine maternity, paternity, or match to another relative – eliminate a suspect – free a falsely imprisoned individual – identify human remains

17. Matching DNA Samples RFLP Matching – Two samples that have the same band pattern are from the same person – No two people will show identical band patterns, except identical twins

18. Paternity and Maternity Determination RFLP Inheritance Matching – Each band in a child’s DNA fingerprint must be present in at least one parent

19. RFLP Limitations Although DNA fingerprinting and the use of RFLP’s is very useful it no longer the preferred method for DNA profiling. – Must have a complete DNA sample – Hard to copy complete DNA samples using PCR – STR analysis is now the preferred

20. Genes and Chromosomes Genes are functional units or segments of DNA that code for proteins Genes are located on chomosomes Humans have 46 chromosomes – Inherit 23 from each parent 22 regular chromosomes (autosomes) 1 sex chromosome (X or Y)

21. Junk DNA The HUMAN GENOME contains non-coding regions called junk DNA – Junk DNA can be found between functional genes many randomly repeated sequences (VNTR’s and STR’s) are located between genes or coding at certain loci (locations) number of randomly repeated sequences varies between individuals at each site

22. VNTR Variable Number of Tandem Repeats Short segments of randomly repeated sequences of junk DNA Located in between genes or coding DNA at specific sites or loci Usually between 9-80 base pairs long Number of repeats at specific sites varies from person to person

23. STR’s Short Tandem Repeats Even shorter segments of randomly repeated Short repeat themselves Usually 3-7 base pairs long – Example: GATA Number of times it repeats itself in a sequence varies from person to person (like VNTR’s)

24. DNA Profiling Using STR’s STR’s within a gene locus (location) are counted – Number of short tandem repeats at each site varies from person to person 13 different gene sites or loci are used in analysis

25. DNA Profiling STR’s PCR is used to amplify or make copies of the DNA sample Genes or Loci are isolated and then cut with a restriction enzyme Segments are separated to determine how many times the STR occurred within the gene locus – Each person has two gene sites for each of the 13 loci Inherit one from each parent – Number of repeats at each site is analyzed and compared Example 12/8, 10/8, or 6/ 4 Preferred Current Method Used In PROFILING Easier to extract, cut, and copy amplify from DNA of individual using PCR Can profile even if there is Deteriated or incomplete DNA sample available

27. 13 CODIS STR’s Probability of 2 People Matching STRAfrican AmericanCaucasian 1D3S1358.094.075 2vWA.063.062 3FGA.033.036 4TH01.109.081 5TPOX.090.195 6CSF1PO.081.112 7D5S818.112.158 8D13S317.136.085 9D7S820.080.065 10D8S1179.082.067 11D21S11.034.039 12D18S51.029.028 13D16S539.070.089

28. Probability and STR Analysis 1)What are the chances of two Caucasian individuals being matches for the STR vWA 2)What are the chances that two African American individuals are matches for the STR’s FGA and TP0X? 3)What are the chances of two Caucasian individuals being matches for the STR’s vWA, TH01, and, D18S51? 4)What are the chances of two African American individuals being matches for the STR’s TH01, FGA, vWA, and D18S51?

29. STR Analysis So how unique is our DNA? The chances of the number of STR’s being exactly the same at each of the 13 sites is… 1/575 trillion for Caucasian Americans 1/900 trillion for African Americans

30. Sex Identification Focus is on the amelogenin gene which is located on the X and Y chromosome – Gene codes for tooth pulp – Gene on Y chromosome is 6 bases shorter on X chromosome than the on the Y Females, XX, show only one band pattern when gene is amplified (copied through PCR) and separated through electrophoresis Males will show 2 band patterns when same process is completed

32. CODIS Combined DNA Index System – 13 STR’s are used for profiling and determining matches Computer Program Contains Database of DNA profiles States are mandated to report … – DNA Profiles of convicted sex offenders and other criminals (convicted of certain crimes) – DNA Profiles of unsolved cases – DNA of missing persons – Law Enforcement Agencies can run DNA from new crime scenes through CODIS to determine possible matches

33. Mitochondrial DNA Mitochondria are cell structures found in all humans Mitochondria are the “powerhouses” of the cell and contain they contain DNA – This is separate from the nuclear DNA of the cell and ONLY contains DNA from ONE PARENT. – mDNA is inherited through the ova (egg) from mother This mDNA of MtDNA is inherited maternally – From the mother – Not as exclusive, DNA is the same as all those maternally related. Siblings, cousins, all persons ect… share common DNA if maternally related. mDNA can often be found in samples or remains that do not contain nuclear DNA – Hair shaft, bones, ect…

34. Collection of Biological Evidence What can DNA be obtained from? – Blood – Semen – Epithelial (Skin) Cells from direct transfer – Sweat (epithelial cells) – Saliva (epithelial cells) Latex Gloves must be used to collect specimens thought to contain DNA – Separate gloves for each piece of evidence Prevents contamination

35. Packaging & Preservation of DNA Evidence Samples must be allowed to dry and/or placed in breathable container – Avoids breakdown of DNA by bacteria A small portion of the sample must be collected and packaged separately, known as substrate control – used for comparative purposes later If does not match then DNA sample is invalid

36. Collection of DNA from Suspects Buccal Swap – Cotton swab used to rub inside of cheek Transfer of epithelial cells to tranfer Specimens must be collected and preserved in same manner previously discussed – Separate gloves each sample – Substrate control

37. DNA Backlog # of cases involving DNA evidence far exceeds the processing abilities of crime labs Many crime labs are backlogged several months Other pieces of evidence, blood ect… are often used first to determine whether to include or exclude a suspect – If other evidence includes suspect then DNA evidence is processed to add INDIVIDUALIZED evidence to the case