DNA Forensics Lab – background DNA A. Human genome contains 3 billion base pairs
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Transcript of DNA Forensics Lab – background DNA A. Human genome contains 3 billion base pairs
DNA Forensics Lab – backgroundI. DNA
A. Human genome contains 3 billion base pairs
B. Human DNA is about 99.9% the sameC. The differences in DNA is concentrated
in specific regions of DNA
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
E. DNA can be found where?1. hair2. skin3. saliva4. semen5. blood (most)6. pretty much in any cells of body
II. DNA FingerprintingA. Defn – a pattern of DNA characteristics that are unique to an individualB. How a DNA fingerprint is made1. Obtain DNA 2. Isolate the VNTR – 2 methodsa. Restriction Enzymes – chemicals cut DNA around each VNTRb. PCR – polymerase chain reaction – use primers to “find” VNTR and make many copies of them
3. Put DNA into a gel a. gel has massive intertwining matrixb. 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 firstii. Like a small snake vs large snake through a jungled. add stain to make DNA bands appear
Gel electrophoresis
V. DNA Forensics Lab EquipmentA. micropipetter
1. used to measure small volumes2. microliter (µl) = 1.0 x10-6 liters
B. gel case – to form and store gel
C. comb – to make wells (holes) in gel
D. electrophoresing chamber – to hold gel and run electricity through gel
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!
III. Restriction Enzymes (RE)A. Like chemical scissors to cut DNA B. Each RE cuts at specific site
1. there are at least 100 RE2. ie:could cut after base sequence
GCATT
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
• Sample match DNA: Which lanes match?
1 2 3 4
• D. Paternity cases are dif– Match dna bands from kid with mom– Left over on kids must be from dad – so match up
• Sample paternity:
ma kid pa 1 pa 2
Bellringer 4/25/11
• 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?
IV. PCR – Polymerase Chain ReactionA. Method to make many copies of small pieces of DNA – from small amount of DNAB. Use primers to find VNTR
OR STRs- Short Tandem Repeats C. The VNTRs are then copied many timesD. idea: heat, cool, heat, cool – at least 20 x
E. PCR Mixture1. primers – to find VNTRs – start
replication2. DNA polymerase (taq) – enzyme to
add nucleotides (replicate)3. buffer – solution4. nucleotides – to build more DNA
F. Steps of PCR1. Denature - heat DNA – to split apart 2. Hybridization or Annealing a. cool DNAb. DNA primers and polymeraseattach to DNA - primers read from 5’ to 3’ end of DNA3. DNA synthesis or Extensiona. still coolb. make copy of short piece of DNA- using DNA polymerase4. repeat cycle – heat/cool
VIDEOS
DNA forensics – United Streaming – 3:30http://player.discoveryeducation.com/index.cfm?guidAssetId=173906D4-98A2-4091-8389-804E995C5A92&blnFromSearch=1&productcode=US#PCRhttp://highered.mcgraw-hill.com/olc/dl/120078/micro15.swfhttp://www.sumanasinc.com/webcontent/animations/content/pcr.html
Electrophoresis – good activityhttp://learn.genetics.utah.edu/content/labs/gel/