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

PCR

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/