DNA: the indispensable forensic science tool

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 • DNA Heated to separate two strands • Add primer and lower temperature -primer = short strand of complementary DNA that will target and attach to original separated strands • Add 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’sRestriction Fragment Length Polymorphisms • Takes advantage of the fact RESTRICTIONENZYMES 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 • 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 • 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 SamplesRFLP • 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 DeterminationRFLP • 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 • Junk DNA even be found within our genes • many randomly repeated sequences (VNTR’s and STR’s) are located within genes or coding , functional DNA sites(loci) • number of randomly repeated sequences varies between individuals at each site

22. VNTRVariable Number of Tandem Repeats • Short segments of randomly repeated sequences of junk DNA • Located within 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’sShort 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 STR African American Caucasian 1 D3S1358 .094 .075 2 vWA .063 .062 3 FGA .033 .0364 TH01 .109 .081 5 TPOX .090 .1956 CSF1PO .081 .112 7 D5S818 .112 .158 8 D13S317 .136 .085 9 D7S820 .080 .065 10 D8S1179 .082 .067 11 D21S11 .034 .039 12 D18S51 .029 .028 13 D16S539 .070 .089

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