1. Laboratory #1: Forensic DNA Fingerprinting
Wheeler High School The Center for Advanced Studies in Science, Math & Technology
Laboratory #1: Forensic DNA Fingerprinting
Lab Timeline
Post-AP DNA/Genetics – Ms. Kelavkar

2. Laboratory Day 1 Restriction Enzyme Digest
Post-AP DNA/Genetics – Kelavkar

3. Laboratory Day 2
Gel Electrophoresis
Post-AP DNA/Genetics – Kelavkar

4. Laboratory Day 3 Destain gels, observe results & analyze
Post-AP DNA/Genetics – Kelavkar

5. Laboratory Day 4 Plasmid Mapping How to guide
Post-AP DNA/Genetics – Kelavkar

6. Day 1: Restriction Enzyme Digest
Wheeler High School The Center for Advanced Studies in Science, Math & Technology
Day 1: Restriction Enzyme Digest
Laboratory #1: Forensic DNA Fingerprinting
Post-AP DNA/Genetics – Kelavkar

7. DNA Review Structure Function O O Phosphate Sugar Phosphate Sugar O O
Base O O
CH2
Sugar O
Phosphate O P O
Base O O
CH2
Sugar OH

8. What are Restriction Enzymes?
Evolved by bacteria to protect against viral infection
“Molecular Scissors”
Cut at specific sites
How was this evolutionary advantageous?
How might bacteria protect their own DNA from these restriction enzymes?

9. How are restriction enzymes named?
Restriction endonucleases
Endo = within
Nuclease = enzyme that cut’s nucleic acids
EcoRI
Common restriction enzyme
Isolated from Escherichia coli
Thus Eco…
The RI specifies the a specific place the enzyme is encoded

10. Blunt vs. Sticky Ends
Blunt end =
Sticky end =

11. Restriction Site Where the restriction enzyme cuts
If a specific restriction site occurs in more than one location on a DNA molecule, cuts will be made at each of those sites, giving multiple fragments.
EcoRI
– Eschericha coli
– 5 prime overhang

12. Enzyme Site Recognition
Restriction site
Palindrome
• Each enzyme digests (cuts) DNA at a specific sequence = restriction site
• Enzymes recognize 4- or 6- base pair, palindromic sequences
(eg GAATTC)
Fragment 2
Fragment 1

13. Why use RE’s when conducting gel electrophoresis?
Used to cut strands of DNA at specific sites
Cut DNA can be separated via gel electrophoresis
Technicians use RFLP (Restriction Fragment Length Polymorphism) to provide unique banding patterns based on restriction sites

14. Today’s Lab Considerations…
Lab Safety
Lab Groups
Read protocol & conduct experiment
Review Questions
Clean-Up & Prep for next class

15. Day 2: Agarose Gel Electrophoresis
Wheeler High School The Center for Advanced Studies in Science, Math & Technology
Day 2: Agarose Gel Electrophoresis
Laboratory #1: Forensic DNA Fingerprinting
Post-AP DNA/Genetics – Ms. Kelavkar

16. Agarose Gel Electrophoresis
Separates DNA fragments by size
Why do we load DNA on the negative end of the chamber?

17. Running Your Gel Agarose gel sieves DNA fragments according to size
Small fragments move farther than large fragments
Why does the density of the gel matter?
Power Supply

18. Staining Gel
DNA is colorless, so we need to stain it to see the fragments
We will use a 1x concentration of Fast Blast DNA stain
Overnight procedure

19. Today’s Lab Considerations…
Lab Safety
Read protocol run gels
Stain gels before the end of class as directed
Review Questions

20. Day 3: Visualization of Gel
Wheeler High School The Center for Advanced Studies in Science, Math & Technology
Day 3: Visualization of Gel
Laboratory #1: Forensic DNA Fingerprinting
Post-AP DNA/Genetics – Ms. Kelavkar

21. Observing Your Gel Trace your gel & take a digital picture
Use light box and plastic wrap
Post-Lab analysis and interpretation (graphing) of results

22. RFLP – Interpreting Results
PstI
EcoRI
CTGCAG
GAGCTC
GAATTC
GTTAAC
Allele 1 1 2 3
CGGCAG
GCGCTC
GAATTC
GTTAAC
Allele 2 3
Fragment 1+2
Different
Base Pairs
No restriction site M
A-1
A-2
Electrophoresis of restriction fragments
M: Marker
A-1: Allele 1 Fragments
A-2: Allele 2 Fragments +

23. Molecular Weight Determination
Fingerprinting Standard Curve: Semi-log
Size (bp) Distance (mm)
23, 9, 6, 4, 2, 2,

24. Today’s Lab Considerations…
Obtain results/trace & picture
Post-Lab thought questions
Complete electrophoresis data table
Graph data
Answer post-lab interpretation of results questions
How to dry gels

25. Day 4: Drawing Restriction Maps
Wheeler High School The Center for Advanced Studies in Science, Math & Technology
Day 4: Drawing Restriction Maps
Laboratory #1: Forensic DNA Fingerprinting
Post-AP DNA/Genetics – Ms. Kelavkar

26. Reading Restriction Maps
Ori (Origin of replication)
Beta lactamase – allows for resistance to ampicillin
Lambda sequence – foreign DNA insert
Post-AP DNA/Genetics – Ms. Kelavkar

27. Mapping Plasmids
This requires logic!