
8fae5eaf179b75b32c8858b073decd30.ppt
- Количество слайдов: 56
DNA: The Indispensable Forensic Science Tool Chapter 13
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 heredity
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 (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 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
Sickle Cell Anemia
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 between bases break New bases attach to “old” bases Two identical DNA molecules produced Polymerases- enzymes
DNA Replication
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
Restriction Enzymes- Cut DNA
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
Gel Electrophoresis
Southern Blotting Transfer of separated DNA fragments on to a nylon membrane
Hybridization Radioactive probes attach to complementary bases for visualization
Hybridization
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
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 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
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- 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- 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 Robotics (< error) Bioanalyzer Electropherogram
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, not 2 (only 1 chromosome) Multiple males
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 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 Time consuming Costly Difficult Less discriminating
DNA Evidence
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 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 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 or rectal cells Table 13 -2 pg. 391
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 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 gloves often Use disposable forceps Package individually
DNA QUIZ http: //investigation. discovery. com/investiga tion/quiz-central/dna-forensics/dnaevidence. html
8fae5eaf179b75b32c8858b073decd30.ppt