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Forensic Science: DNA

Forensic Science: DNA. Prof. J. T. Spencer. DNA. Deoxyribonucleic Acid What are nucleic Acids Structure of DNA DNA vs. RNA “Blue print” of life How it “transcribes” life Where is it located Forensic Applications. DNA. Timeline of DNA 1868 Miescher “discovers” DNA

norman-knowles
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Forensic Science: DNA

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  1. Forensic Science: DNA Prof. J. T. Spencer

  2. DNA • Deoxyribonucleic Acid • What are nucleic Acids • Structure of DNA • DNA vs. RNA • “Blue print” of life • How it “transcribes” life • Where is it located • Forensic Applications

  3. DNA Timeline of DNA 1868 Miescher “discovers” DNA 1953 Watson and Crick report double helix structure 1977 First human gene cloned. 1985 Jeffreys reports VNTR DNA sequences 1985 First report of PCR method 1986 Jeffreys uses DNA to solve first murder case (Picthfork case) 1987 First conviction on DNA evidence (Andrews case) 1991 STRs first reported 1998 FBI starts CODIS database 2005 2.5 million DNA “finger-prints” in FBI database

  4. Nucleic Acids • Nucleic Acids - chemical carriers of genetic information (DNA and RNA). • Consist of; • A phosphoric Acid Molecule (H3PO4) • A five carbon Sugar (ribose) • A nitrogen containing base DEOXYRIBOSE RIBOSE

  5. Nucleic Acids Three “Building Blocks” linked through condensation reactions to form polymers. Nucleotide - unit of the three building blocks (phosphate, sugar, and base) Phosphate Base Nucleotide Sugar

  6. Nucleic Acids: Bases Guanine Adenine Cytosine Thymine (DNA Only) Uracil (RNA Only)

  7. Phosphate Base Sugar Phosphate Base Sugar Phosphate Base Sugar Phosphate Nucleic Acids • Nucleic Acid Polymer (i.e., DNA) Polymer

  8. Nucleic Acids • Nucleic Acid Polymer (i.e., DNA)

  9. Nucleic Acids • Double Helix - Hydrogen Bonds

  10. Nucleic Acids Double Helix - Hydrogen Bonds physics.bu.edu/~neto/ Topic6.htm

  11. Base Pairs • Hydrogen Bonding

  12. DNA Structure Major Groove Minor Groove

  13. DNA Structure

  14. DNA Structure

  15. chromosome cell nucleus Double stranded DNA molecule Individual nucleotides DNA in the Cell

  16. DNA Structure • (1) Information in the order of nucleotides in DNA can be transcribed and translated to direct the preparation of proteins in the cell. • (2) Every three nucleotides in DNA codes for one amino acid in the formation of a protein. • (3) Parts of DNA coding for proteins are called genes. • (4) DNA contains very large parts that code for nothing known.

  17. DNA • Nuclear DNA • Maternal DNA • Paternal DNA Chromosome DNA

  18. DNA: Chromosomes

  19. Human Genome Project • (1) Completed in 2003. • (2) Determined base (nucleotide) sequence of all 30,000 human genes (the order of xx million nucleotides). • (3) Found humans share ca. 93% of our DNA code with roundworms. • (4) Found only ca. 1.5% of our DNA codes for compounds.

  20. DNA • Transcription: • 3 Base pairs corresponds to 1 amino acid. • Builds Proteins • Proteins - enzymes, all functions for life.

  21. DNA Transcription • Transcription:

  22. DNA Cellular Sources • Nuclear DNA • Located in nucleus. • inherited 1/2 from mother and 1/2 from father. • Mitochondrial DNA • Located in mitochondria (cellular respiration). • inherited solely from mother.

  23. Where is DNA Nuclear DNA Mitochondrial DNA • Cellular Matter • Organ Cells • Hair Roots • Sweat • Semen • Blood (not RBCs) • It’s the same from every source in the body. • Usually in the cell nucleus.

  24. Where DNA Samples found: Blood Stains Semen Stains Chewing Gum Stamps & Envelopes Plant Material Sweaty Clothing Bone Hair (sheath) Fingernail Scraping Saliva Animal Material Other

  25. DNA Evidence

  26. Mitochondrial DNA • Lines of evidence independently suggest that Africa is the birthplace of humankind. (East Africa or southern Africa) • By examining DNA in living people, the origin of our species est. between 100 000 - 150 000 years before the present. • The use of mtDNA and the non-recombining portion of the Y chromosome have shown how females and males, respectively, have contributed to the gene pool of southern African populations. • It is possible to reconstruct the history of mutations to a common ancestor that would have lived in prehistoric times..

  27. Human Identity Testing • Forensic cases - Match or eliminate suspect with evidence. • Paternity testing - Match or eliminate possible father(s). • Historical investigations - Czar Nicholas and Royal family. • Missing persons investigations - Partial remains with known samples. • Mass disasters - Identifying remains. • Military DNA Uses - Records of personnel (DNA “dog tags”), identifying combatants, identifying wanted terrorist suspects. • Convicted felon DNA databases - NYS and FBI databases.

  28. DNA Analysis • Collection of Sample • Separation and Purification of DNA • Amplification (making copies of DNA) • Cutting/Separation of Fragments • Comparison and Analysis

  29. DNA “Fingerprinting” • DNA used to identify individuals • Spacing between genes is different for each individual • Collect DNA at crime scene (both evidence and suspect DNA) • Make more DNA using “PCR” (Polymerase Chain Reaction to “amplify” DNA) - no longer need a big sample - can be done from 1 DNA piece. • Cut DNA with “restriction” Enzyme (next slide) • Separate DNA fragments using “gel” • Detect DNA (25- 60 bands on gel ) 1”:1,000,000 have same pattern

  30. Tandem Repeats • DNA forms genes that code for proteins. • Not all DNA codes for proteins (past useless info separating genes). • Intergene regions contain many A,T,C,G repeats. (why?? Unknown - maybe spacers maybe discarded genetic info - about 30% of DNA code). called Tandem Repeats. • Forensic DNA typing uses tandem repeats. • All humans have many tandem repeats. • Great variation per person, however, in how many and where these repeats occur.

  31. Tandem Repeats • Tandem Repeats. • Natural selection eliminates mutations that result in non- or less viable phenotypes. The 95% of the DNA that does not encode protein is much less subject to natural selection, and the noncoding DNA has far more sequence variation (per person). • Variable Number Tandem repeats (VNTR)

  32. Tandem Repeats • Short Tandem Repeats.

  33. Insulin Gene Insulin Cut out gene Plasmid Circular DNA Cut Insert Insulin DNA Remove DNA Plasmid Bacteria Recombinant DNA (Cutting) • Therapeutic Proteins and other useful substances from other organisms. • Splicing and recombining genes • Gene retrieved from one organism can be reproduced identically (cloned) in bacteria: Insulin; “Flounder genes into a Strawberry” - flounder temperature resistance into temperature sensitive strawberries

  34. Gene Therapy • Gene therapy replaces missing or damaged proteins by providing the gene that will direct its manufacture. This involves stuffing the gene into a viral carrier (vector) then injecting the loaded beneficial virus into the patient. Once inside the cells, the genetic material is released and begins making the protein that should cure the disease or at least alleviate symptoms. • Cures for several problems found in 2000; • Hemophilia • Severe combined immunodeficiency disease, or SCID • Squamous cell carcinoma (enhances tumor necrosis factor) • using DNA to carry a substance that stimulates blood vessel growth to damaged heart tissue.

  35. DNA “Fingerprinting” RFLP • Restriction enzyme cuts at ….AAGCTT….. • Spacing between cutting sites for enzyme is different for different individuals

  36. Person A Person B 4 3 5 1 2 6 DNA DNA Cut sites 4 1 5 2 6 3 Separate by Gel Electrophoresis Different DNA lengths 1, 2, 3 not equal to 4, 5, 6 DNA “Fingerprinting” • Restriction enzyme cuts at ….AAGCTT….. • Spacing between cutting sites for enzyme is different for different individuals

  37. DNA- - + DNA- DNA- DNA- DNA- DNA Separation Mechanism • Size based separation due to interaction of DNA molecules with entangled polymer strands • Polymers are not cross-linked (as in slab gels) • “Gel” is not attached to the capillary wall • Polymer length and concentration determine the separation characteristics

  38. Gel Electrophoresis (2) Spot on Gel (1) Amplify DNA sample, react with restriction enzymes, prepare sample (3) Use elec. Current to move DNA pieces (DNA negative, smaller pieces travel fastest, detect using radioactivity)

  39. Gel Electrophoresis Lanes 1-3 contain DNA samples, perhaps from the same stock of DNA digested with 3 different Restriction Enzymes. Lanes 4 and 5 are called Standard Lanes, or sometimes Molecular Weight Markers or "Ladders". They are similar to the Control in an experiment, because we know exactly how they are going to turn out every time. These lanes contain DNA of a specific, predetermined size from a known source, digested with a Restriction Enzyme that cuts that piece of DNA a known number of times, yielding a predicted number of bands whose size we know exactly. For example,Lane 5 is DNA from the E. coli bacteriophage Lambda, digested with Hin dIII. We know exactly how long each of the visible fragments are.

  40. RFLP Analysis • Enzymes break DNA into restriction fragments • Measurements taken of fragments that vary in length bet’n people (length polymorphism) • Can produce extremely low random match probabilities • Requires relatively large fresh samples (>50 ng DNA) • Slow and expensive

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