Chapter 9 DNA The Indispensible Forensic Science Tool

1. Chapter 9 DNA The Indispensible Forensic Science Tool Learn the parts of a nucleotide and know how they are linked together to make DNA. Understand how DNA strands coil into a double helix. Describe the concept of base pairing as it relates to the double helix structure of DNA. Explain how the sequence of bases along a DNA strand ultimately determines the structure of proteins that are synthesized within the body. Describe how a double-strand DNA replicates itself. What are the implications of this process for forensic science? Created by C. Ippolito April 2005

2. DNA Created by C. Ippolito April 2005

3. The Structure of DNA • DNA is composed of nucleotides • Each DNA nucleotide has: • Sugar – deoxyribose • Phosphate Group • Nitrogen Base • Purines – double ring – adenine & guanine • Pyrimidines – single ring – thymine & cytosine Created by C. Ippolito April 2005

4. DNA Nucleotides • Chargaff’s Rule • % of guanine always equals amount of cytosine • % of adenine always equals amount of thymine • G = C • A = T Created by C. Ippolito April 2005

5. X-Ray Evidence • Rosalind Franklin • used x-ray crystallography to photograph DNA • Photograph 51 used by Watson and Crick to build model • Visit these links to find out more about Miss Franklin and her photo http://www.pbs.org/wgbh/nova/photo51/http://www.providencepictures.com/pp/press_photo51_globeandmail.html Created by C. Ippolito April 2005

6. Watson and Crick Model • Shape is a “twisted ladder” or Double Helix • Sides - sugar and phosphates • Steps – nitrogen base pairs • adenine and thymine OR guanine and cytosine • Link for interactive tutorial: • http://www2.mrc-lmb.cam.ac.uk/DNA/index.html • To use site download Chime Plug-In from mr-ippolito-science-class.org Created by C. Ippolito April 2005

7. Chromosomes and DNA Replication Created by C. Ippolito April 2005

8. DNA and Chromosomes • Chromosomes are made of DNA & Proteins • DNA is wrapped around the protein histone • Nucleosomes are groups of histones Created by C. Ippolito April 2005

9. DNA Replication • DNA makes a copy of itself before cell division • DNA separates into two strands • Each strand acts as a template • Complementary strands formed by base pairing • Catalyzed by the enzyme DNA polymerase visit these links: http://academy.d20.co.edu/kadets/lundberg/DNA_animations/DNAreplication.mov http://academy.d20.co.edu/kadets/lundberg/DNA_animations/dna.dcr Created by C. Ippolito April 2005

10. DNA Unzips • a special enzyme opens the double helix • It does this by breaking the weak hydrogen bonds between bases • The enzyme slowly proceeds down the middle of the DNA molecule • This will allow exposed bases to be matched forming new “sides” • until the DNA molecule is completed opened Created by C. Ippolito April 2005

11. The enzyme positions them opposite the proper base to form new base pairs • Each strand is half new and half old DNA • New hydrogen bonds form between the bases • Nucleotides from cell storage area are brought in by enzymes • Resulting in two identical strands of DNA DNA Polymerase Created by C. Ippolito April 2005

12. RNA and Protein Synthesis Created by C. Ippolito April 2005

13. Structure of RNA • Each RNA nucleotide has: • Sugar –ribose • Phosphate Group • Nitrogen Base • Purines – double ring – adenine & guanine • Pyrimidines – single ring – uracil & cytosine Created by C. Ippolito April 2005

14. Types of RNA • Three kinds of RNA • Messenger RNA (mRNA) – carries instructions to ribosome • Transfer RNA (tRNA) – carries amino acids to ribosome • Ribosomal RNA (rRNA) – forms part of the ribosome Created by C. Ippolito April 2005

15. Transcription • DNA copies its nucleotide sequence into RNA • RNA polymerase binds to DNA promoter region. • DNA separates into two strands. • RNA polymerase uses one DNA strand as a template. • Complimentary bases match template to form RNA. • RNA leaves and DNA closes Created by C. Ippolito April 2005

16. RNA Editing • Introns – parts of DNA that form pre-edited RNA that are cut out of the mRNA • Exons – parts of DNA that form parts of pre-edited RNA that are spliced together to make mRNA. Created by C. Ippolito April 2005

17. Genetic Code • Nitrogen base sequence “names” amino acids of a protein • 20 different amino acids • 4 bases A C G U • 3 bases names an amino acid (codon) Created by C. Ippolito April 2005

18. Translation • mRNA decoded at ribosome to form proteins • mRNA attaches to ribosome • tRNA anticodon matches mRNA codons • Amino acids on tRNA’s bond together • Ribosome moves to next repeating process • At end mRNA, protein and ribosome released • Visit: Lew-Port’s Biology Place - Protein Synthesis Animation • Biostudio Protein Synthesis Animation Created by C. Ippolito April 2005

19. Polymerase Chain Reaction • lab technique used to “amplify” small amounts • uses Taq polymerase derived from hot springs bacteria • in a few hours 30 cycles results billion fold increase • for more information visit: • http://www.sumanasinc.com/webcontent/anisamples/molecularbiology/pcr.html Created by C. Ippolito April 2005

20. Recombinant DNA (rDNA) • inserts genomic DNA from one species into bacterial plasmids • uses restriction enzymes to remove specialized sections of DNA • once in bacterium “product” coded for by DNA can be identified • used to produce • human growth hormone • insulin Created by C. Ippolito April 2005

21. DNA Tandem Repeats • DNA sequences that are found in multiple copies in human DNA • seem to act as “fillers” or “spaces” between coding regions • allow forensic scientists to distinguish individuals through DNA Typing Created by C. Ippolito April 2005

22. Restriction Fragment Length Polymorphisms • are repeating DNA sequences that can be removed by restriction enzymes • length differences in these sequences can be used to distinguish individuals • sequences are separated by gel electrophoresis • Southern blotting transfers separated fragments to nylon membrane • Hybridization attaches radioactive compliments to strands • Radioactive material “develops” film to form “picture” of DNA fragments Created by C. Ippolito April 2005

23. Short Tandem Repeats • newer DNA profiling technique • needs less DNA because sequences are smaller • multiple repeats of same bases • THO1 – A-A-T-G repeats • PCR amplifies the STRs • Multiplexing • allows multiple different STRs to be identified allowing “narrowing” identification in population • 13 different CODIS STRs are found with different frequencies within the population Created by C. Ippolito April 2005

24. Capillary Electrophoresis • allows automation / shortens time • thin glass column coated with gel immersed in buffer • DNA injected in one end • as STRs move through gel a detector tracks them to form a electropherogram Created by C. Ippolito April 2005

25. Sex Identification with STRs • amelogenin gene • controls formation of tooth pulp • on both the X chromosome and Y chromosome • X version six (6) bases shorter than Y version • females with XX genotype show one band • males with XY genotype show two bands • 20 different Y-STR markers identified • can distinguish between males in sexual assaults Created by C. Ippolito April 2005

26. Mitochondrial DNA • Mitochondria • powerhouse of cell – site of aerobic respiration • has own DNA – mtDNA • only maternal DNA • mtDNA – circular chromosome of some 17,000 bases – HV1 and HV2 regions used • used when no nuclear DNA available • used to ID remains (skeletal) • need reference samples from female family members Created by C. Ippolito April 2005

27. Collect Biological Evidence for DNA Analysis • STR profiling allows typing with as few as 125 picograms of DNA • picogram = 10-12 gram or .000000000001 gram • 7 picograms of DNA in a human cell • as little as 18 cells can allow a valid STR profile to be developed • goal to get profile from ONE (1) human cell Created by C. Ippolito April 2005

28. Common Sources of DNA Created by C. Ippolito April 2005

29. Proper Collection • DNA is in body fluids – infectious • disposable gloves • change often • face masks • shoe covers • overalls • must prevent “cross contamination” Created by C. Ippolito April 2005

30. Packaging Biological Evidence • moisture grows bacteria and mold • always in paper or well ventilated box • swabs need to be air-dried for 5-10 minutes before boxing • cool storage out of sunlight • exception: blood in soil must be FROZEN in glass/plastic • substrate control • sample of unstained surface material for comparison Created by C. Ippolito April 2005

31. Reference DNA Specimens • DNA in samples compared to victims and suspects • 7 ml blood collected with EDTA (inhibits enzyme degradation) in sterile vacuum tubes and refrigerated • buccal swabs – cheek cells for sample Created by C. Ippolito April 2005

32. Contamination of DNA Evidence • as simple as a cough or a sneeze • prevent • change gloves with each sample • substrate control for each sample • disposable forceps for each small item (cigarette butts, licked stamps, drinking straws, etc. • each in own ventilated container Created by C. Ippolito April 2005