DNA Forensics Lab – background DNA A. Human genome contains 3 billion base pairs

1. DNA Forensics Lab – background DNA A. Human genome contains 3 billion base pairs B. Human DNA is about 99.9% the same C. The differences in DNA is concentrated in specific regions of DNA

2. D. Variable Number of Tandem Repeats (VNTR) 1. DNA sequences from 3 to 30 base pairs (bp) long 2. Each person has unique position and number of the VNTRs in all DNA 3. From these dif a DNA fingerprint can be made – to distinguish the dif

3. E. DNA can be found where? 1. hair 2. skin 3. saliva 4. semen 5. blood (most) 6. pretty much in any cells of body

4. II. DNA Fingerprinting A. Defn – a pattern of DNA characteristics that are unique to an individual B. How a DNA fingerprint is made 1. Obtain DNA 2. Isolate the VNTR – 2 methods a. Restriction Enzymes – chemicals cut DNA around each VNTR b. PCR – polymerase chain reaction – use primers to “find” VNTR and make many copies of them

6. 3. Put DNA into a gel a. gel has massive intertwining matrix b. DNA is negative charge – so can run electricity through gel (DNA will travel to positive) c. DNA will separate by size (VNTR) i. short pieces out first ii. Like a small snake vs large snake through a jungle d. add stain to make DNA bands appear

7. Gel electrophoresis

8. V. DNA Forensics Lab Equipment A. micropipetter 1. used to measure small volumes 2. microliter (µl) = 1.0 x10-6 liters B. gel case – to form and store gel

9. C. comb – to make wells (holes) in gel D. electrophoresing chamber – to hold gel and run electricity through gel

10. E. Power Source – supplies energy for electricity F. Centrifuge – to spin microtubes really fast to move all sample to bottom of tube  must be safe – balanced!

11. III. Restriction Enzymes (RE) A. Like chemical scissors to cut DNA B. Each RE cuts at specific site 1. there are at least 100 RE 2. ie:could cut after base sequence GCATT

12. Reading a DNA Fingerprint • A. The fingerprint is all the bands together • B. recall: shortest pieces are away from gel • C. you can match evidence to suspect

13. Sample match DNA: Which lanes match? 1 2 3 4

14. D. Paternity cases are dif • Match dna bands from kid with mom • Left over on kids must be from dad – so match up

15. Sample paternity: ma kid pa 1 pa 2

16. Bellringer4/25/11 • 1. What is a VNTR? Why are they important in DNA fingerprinting? • 2. What are restriction enzymes? • 3. Do you put wells on negative or positive side of electrophoresing chamber, why? • 4. What does a centrifuge do?

17. IV. PCR – Polymerase Chain Reaction A. Method to make many copies of small pieces of DNA – from small amount of DNA B. Use primers to find VNTR OR STRs- Short Tandem Repeats C. The VNTRs are then copied many times D. idea: heat, cool, heat, cool – at least 20 x

18. E. PCR Mixture 1. primers – to find VNTRs – start replication 2. DNA polymerase (taq) – enzyme to add nucleotides (replicate) 3. buffer – solution 4. nucleotides – to build more DNA

19. F. Steps of PCR 1. Denature - heat DNA – to split apart 2. Hybridization or Annealing a. cool DNA b. DNA primers and polymerase attach to DNA - primers read from 5’ to 3’ end of DNA 3. DNA synthesis or Extension a. still cool b. make copy of short piece of DNA - using DNA polymerase 4. repeat cycle – heat/cool

20. PCR

21. VIDEOS DNA forensics – United Streaming – 3:30 http://player.discoveryeducation.com/index.cfm?guidAssetId=173906D4-98A2-4091-8389-804E995C5A92&blnFromSearch=1&productcode=US# PCR http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf http://www.sumanasinc.com/webcontent/animations/content/pcr.html Electrophoresis – good activity http://learn.genetics.utah.edu/content/labs/gel/