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DNA: The Indispensable Forensic Science Tool Chapter 13 DNA: The Indispensable Forensic Science Tool Chapter 13

DNA Deoxyribonucleic Acid 1985 - Dr. Alec Jeffreys- Leicester University DNA Fingerprinting- isolating and DNA Deoxyribonucleic Acid 1985 - Dr. Alec Jeffreys- Leicester University DNA Fingerprinting- isolating and reading DNA markers DNA profiling/ DNA typing Individualization of biological evidence (blood, semen, hair, tissue)

DNA 60 trillion cells Chromosomes (46, 23 pairs) Genes (30, 000)- fundamental unit of DNA 60 trillion cells Chromosomes (46, 23 pairs) Genes (30, 000)- fundamental unit of heredity

DNA 1953 - Watson and Crick, Franklin DNA Structure- double helix, twisted ladder Polymer DNA 1953 - Watson and Crick, Franklin DNA Structure- double helix, twisted ladder Polymer composed of nucleotides (building blocks)

DNA Nucleotide contains three subunits: 1 - simple sugar- deoxyribose 2 - phosphate group DNA Nucleotide contains three subunits: 1 - simple sugar- deoxyribose 2 - phosphate group (phosphorus containing) phosphate group + deoxyribose (sides of ladder, backbone) 3 - complementary nitrogen bases (rungs) held together w/ weak hydrogen bonds adenine - thymine cytosine – guanine 100 million pairs/chromosome 3 billion/cell

DNA DNA

DNA at Work DNA controls cell’s activities by directing amounts and kinds of protein DNA at Work DNA controls cell’s activities by directing amounts and kinds of protein molecules the cell will produce Sequence of nucleotides determines what amino acids will be used to build a protein molecule Triplet- 3 DNA bases wh/code for a specific amino acid Twenty AA Thousands of proteins

DNA at Work DNA at Work

Sickle Cell Anemia Sickle Cell Anemia

Human Genome Project 1987 -Dept. of Health & National Institutes of Health Genome- all Human Genome Project 1987 -Dept. of Health & National Institutes of Health Genome- all the genetic material in the chromosomes of an organism “Mapping” DNA patterns Inherited traits Genetic diseases- Huntington’s disease- 4 Alzheimer’s disease-14, 19, 21 Evolution

DNA Replication DNA makes identical copy of itself during interphase DNA “unzips” when bonds DNA Replication DNA makes identical copy of itself during interphase DNA “unzips” when bonds between bases break New bases attach to “old” bases Two identical DNA molecules produced Polymerases- enzymes

DNA Replication DNA Replication

PCR- Polymerase Chain Reaction Laboratory DNA replication Small amount of DNA can yield millions PCR- Polymerase Chain Reaction Laboratory DNA replication Small amount of DNA can yield millions of copies DNA Thermal Cycler (two minute cycles) More about this later

Recombinant DNA Ex- Insulin, Growth Hormone Recombinant DNA Ex- Insulin, Growth Hormone

Restriction Enzymes- Cut DNA Restriction Enzymes- Cut DNA

DNA Typing- RFLP Restriction Fragment Length Polymorphisms Tandem Repeats- part of DNA molecule where DNA Typing- RFLP Restriction Fragment Length Polymorphisms Tandem Repeats- part of DNA molecule where a nucleic acid base sequence repeats itself over and over ( >30% “junk”) Spacers between coding regions of DNA 15 -30 bases repeating up to 1000 times

RFLP RFLP

Gel Electrophoresis Gel Electrophoresis

Southern Blotting Transfer of separated DNA fragments on to a nylon membrane Southern Blotting Transfer of separated DNA fragments on to a nylon membrane

Hybridization Radioactive probes attach to complementary bases for visualization Hybridization Radioactive probes attach to complementary bases for visualization

Hybridization Hybridization

Washing and Autoradiography Unbound probes are removed and nylon sheet is exposed to X-ray Washing and Autoradiography Unbound probes are removed and nylon sheet is exposed to X-ray film Autoradiogram of RFLP is produced Resulting bands are compared

Monica Lewinsky’s Dress Monica Lewinsky’s Dress

Polymerase Chain Reaction Multiplies DNA strands (< 200 bases) Heating cooling strand rebuilding Two Polymerase Chain Reaction Multiplies DNA strands (< 200 bases) Heating cooling strand rebuilding Two minute cycles 25 -30 cycles 1, 000 copies One-billionth of a gram DNA Utilizes shorter strands- more stable, less degradation

PCR Step 1 - Separate strands- 94°C Step 2 - Primers (short strand of PCR Step 1 - Separate strands- 94°C Step 2 - Primers (short strand of DNA used to target both sides of a DNA region for replication) hybridize w/DNA strands (lower temp) Step 3 - DNA polymerase + Free nucleotides (72°C) DNA molecule rebuilds 2 complete pairs

PCR PCR

Short Tandem Repeats- STR Area of DNA th/contain short (3 -7 bases) repeating sequence Short Tandem Repeats- STR Area of DNA th/contain short (3 -7 bases) repeating sequence elements (base pairs) < 400 bases Less degradation Recoverable from decomposed bodies or stains Hundreds of STRs in human genes Multiplexing- Several STRs extracted and amplified

STR STR

STR- Product Rule D 3 S 1358 + v. WA + FGA = 1: STR- Product Rule D 3 S 1358 + v. WA + FGA = 1: 5000. . + TH 01 + TPOX + CSF 1 PO =1: 2 mil …+ D 5 S 81+ D 13 S 317 + D 7 S 820 =1: 1 bil …+D 8 S 1179+D 21 S 11+D 18 S 51+D 16 S 539 = 1: 575 Trillion (Caucasians) /1: 900 Trill (African Americans)

STR STR

STR- Capillary Electrophoresis Thin glass column Reduced analysis time Automated sampling and data collection STR- Capillary Electrophoresis Thin glass column Reduced analysis time Automated sampling and data collection

Lab-on–a-Chip Technology Microfluidic Electrophoresis DNA Chip Electrophoresis High Resolution, Accuracy, Reproducibility, Minimal Sample Size Lab-on–a-Chip Technology Microfluidic Electrophoresis DNA Chip Electrophoresis High Resolution, Accuracy, Reproducibility, Minimal Sample Size Robotics (< error) Bioanalyzer Electropherogram

STR- Amelogenin Gene Gender determination Tooth pulp gene X chromosome 1 band Y chromosome STR- Amelogenin Gene Gender determination Tooth pulp gene X chromosome 1 band Y chromosome 2 bands

STR: Y-STR Markers (20) on Y chromosome (male) Characterization of 6 Shows 1 band, STR: Y-STR Markers (20) on Y chromosome (male) Characterization of 6 Shows 1 band, not 2 (only 1 chromosome) Multiple males

Mitochondrial DNA (mt. DNA) Found in mitochondria Inherited from mother Loops of DNA Thirty Mitochondrial DNA (mt. DNA) Found in mitochondria Inherited from mother Loops of DNA Thirty seven genes ~16, 569 bases

mt. DNA Enhanced sensitivity Charred remains, hair shafts, skeletal remains, degraded samples mt. DNA Enhanced sensitivity Charred remains, hair shafts, skeletal remains, degraded samples

mt. DNA Hypervariable region I (HV 1) Hypervariable region II (HV 2) PCR Sequencing- mt. DNA Hypervariable region I (HV 1) Hypervariable region II (HV 2) PCR Sequencing- order of bases mt. DNA population database- HV 1 & HV 2 base sequences (~5000 sequences) 1996 - Tennessee v. Paul Ware (Hair) 1998 - First Lt. Michael J. Blassie

mt. DNA mt. DNA

mt. DNA Time consuming Costly Difficult Less discriminating mt. DNA Time consuming Costly Difficult Less discriminating

DNA Evidence DNA Evidence

Combined DNA Index System CODIS National system of shared databases Convicted offenders, crime scene Combined DNA Index System CODIS National system of shared databases Convicted offenders, crime scene evidence, missing persons Software enables local, state & national crime labs Feb 2007: 4, 907, 077 profiles 45, 400 hits/46, 300 investigations

DNA Evidence Essential, basic, impartial, vital Violent crimes, sex offenses ~ 25% of FBI DNA Evidence Essential, basic, impartial, vital Violent crimes, sex offenses ~ 25% of FBI DNA exams since 1989 have excluded suspects Innocence Projectexonerating wrongly convicted

DNA Evidence STR- need ~250 picograms Human cell- 7 picograms of DNA ~36 DNA-bearing DNA Evidence STR- need ~250 picograms Human cell- 7 picograms of DNA ~36 DNA-bearing cells Low Copy Number- < 36 cells, < normal detection level (18, 9)

DNA Evidence Saliva, blood, semen, urine, skin, ear wax, tissue, hair, dandruff, sweat, vaginal DNA Evidence Saliva, blood, semen, urine, skin, ear wax, tissue, hair, dandruff, sweat, vaginal or rectal cells Table 13 -2 pg. 391

DNA Evidence Photograph biological evidence Minimal personal contact w/evidenceassume infectious (latex gloves, face masks, DNA Evidence Photograph biological evidence Minimal personal contact w/evidenceassume infectious (latex gloves, face masks, shoe covers, coveralls) Clothing, rags, towels, floor cracks Package in paper bag, box , swab container (well-ventilated)-moisture attracts bacteria- refrigerate Soil + blood- glass or plastic & frozen

DNA Evidence Substrate control Known samples from victims/suspects Standard/Reference samples- ~ 7 ml whole DNA Evidence Substrate control Known samples from victims/suspects Standard/Reference samples- ~ 7 ml whole blood (EDTA, refrigerated) Buccal swab Toothbrush, hair brush, razor, dirty laundry, cigarette butts, earplugs

DNA Evidence AVOID CONTAMINATION (shows extra bands) Coughing, sneezing, touching items, incorrect packaging Change DNA Evidence AVOID CONTAMINATION (shows extra bands) Coughing, sneezing, touching items, incorrect packaging Change gloves often Use disposable forceps Package individually

DNA QUIZ http: //investigation. discovery. com/investiga tion/quiz-central/dna-forensics/dnaevidence. html DNA QUIZ http: //investigation. discovery. com/investiga tion/quiz-central/dna-forensics/dnaevidence. html