1. Investigation 9 BIOTECHNOLOGY: RESTRICTION ENZYME ANALYSIS OF DNA
Investigation 9 BIOTECHNOLOGY: RESTRICTION ENZYME ANALYSIS OF DNA* How can we use genetic information to identify and profile individuals?
Pioneering DNA Forensics
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2. Learning targets for Restriction Digestion and Analysis of Lambda DNA lab?
Understand the use of restriction enzymes as biotechnology tools
Become familiar with principals and techniques of agarose gel electrophoresis
Generate a standard curve from a series of DNA size fragments
Estimate DNA fragment sizes from agarose gel data

3. learn how to use restriction enzymes and gel electrophoresis to create genetic profiles.
use these profiles to help Marcus and Laurel narrow the list of suspects in the disappearance of Ms. Mason.

4. Forensic DNA Fingerprinting: Using Restriction Enzymes

5. CSI Scenario:

6. BACKGROUND-page 112
Applications of DNA profiling extend beyond what we see on television crime shows.
Are you sure that the hamburger you recently ate at the local fast-food restaurant was actually made from pure beef? DNA typing has revealed that often “hamburger” meat is a mixture of pork and other non beef meats, and some fast-food chains admit to adding soybeans to their “meat” products as protein fillers. In addition to confirming what you ate for lunch.
DNA technology can be used to determine paternity, diagnose an inherited illness, and solve historical mysteries, such as the identity of the formerly anonymous individual buried at the Tomb of the Unknown Soldier in Washington, D.C.

7. DNA testing also makes it possible to profile ourselves genetically — which raises questions, including Who owns your DNA and the information it carries? This is not just a hypothetical question. The fate of dozens of companies, hundreds of patents, and billions of dollars’ worth of research and development money depend on the answer.
Biotechnology makes it possible for humans to engineer heritable changes in DNA, and this investigation provides an opportunity for you to explore the ethical, social, and medical issues surrounding the manipulation of genetic information.

8. DNA Fingerprinting Real World Applications
• Crime scene
• Human relatedness
• Paternity
• Animal relatedness
Anthropology studies
Disease-causing organisms
Food identification
Human remains
Monitoring transplants

9. The Forensic DNA Fingerprinting concepts:
• DNA structure
• DNA restriction analysis (RFLP)
Agarose gel electrophoresis
Molecular weight determination
Simulation of DNA Fingerprinting
Plasmid mapping

11. DNA Fingerprinting Procedure Overview

12. Lab Time Line • Restriction digest of DNA samples
Introduction to DNA Fingerprinting and RFLP analysis
Electrophoresis on Agarose gels
Analysis and interpretation of results

13. DNA Fingerprinting Procedures Day One

14. What is needed for restriction digestion?
Template DNA, uncut DNA, often bacterial phage DNA
DNA standard or marker, a restriction enzyme of known fragment sizes
Restriction enzyme(s), to cut template DNA
Restriction Buffer, to provide optimal conditions for digestion

15. The DNA Digestion Reaction
Restriction Buffer provides optimal conditions
• NaCI provides the correct ionic strength
• Tris-HCI provides the proper pH
• Mg2+ is an enzyme co-factor

16. DNA Digestion Temperature
Why incubate at 37°C?
• Body temperature is optimal for these and most other enzymes
What happens if the temperature is too hot or cool?
• Too hot = enzyme may be denatured (killed)
• Too cool = enzyme activity lowered, requiring
longer digestion time

18. DNA Restriction Enzymes
• Evolved by bacteria to protect against viral DNA infection
• Endonucleases = cleave within DNA strands
• Over 3,000 known enzymes

19. How does it work? Enzyme Site Recognition Unambiguous Ambiguous
Each enzyme digests (cuts) DNA at a specific sequence  restriction site
Enzymes recognize 4-, 6- or 8- base pair, palindromic sequences
Isoschizomers recognize identical sequences, but have different optimum reaction conditions and stabilities
Can be unambiguous or ambiguous
Unambiguous
Ambiguous

20. 5 vs 3 Prime Overhang
Enzyme cuts
• Generates 5 prime overhang

21. Common Restriction Enzymes
5’ GAATTC 3’
3’ CTTAAG 5’
EcoRI
Escherichia coli
5’ overhang
HindIII
Haemophilus influensae
PstI
Providencia stuartii
3’ overhang
5’ AAGCTT 3’
3’ TTCGAA 5’
5’ CTGCAG 3’
3’ CACGTC 5’

22. 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

23. ■■Activity I: Restriction Enzymes
Read intro to activity 1.
1. What is the sequence of the complementary DNA strand? Draw it directly below the strand.
5’-AAAGTCGCTGGAATTCACTGCATCGAATTCCCGGGGCTATATATGGAATTCGA-3’
3’-TTTCAGCGACCTTAAGTGACGTAGCGTAAGGGCCCCGATATATACCTTAAGCT-5’
2. Assume you cut this fragment with the restriction enzyme EcoRI. The restriction site for EcoRI is 5’-GAATTC-3’, and the enzyme makes a staggered (“sticky end”) cut between G and A on both strands of the DNA molecule. Based on this information, draw an illustration showing how the DNA fragment is cut by EcoRI and the resulting products.
5’-AAAGTCGCTGGAATTCACTGCATCGAATTCCCGGGGCTATATATGG AATTCGA-3’
3’-TTTCAGCGACCTTAAGTGACGTAGCGTAAGGGCCCCGATATATACCTTAA GCT-5’
5’ GAATTC 3’
3’ CTTAAG 5’

26. Restriction Fragment Length Polymorphism RFLP
PstI
EcoRI
Restriction Fragment Length Polymorphism RFLP
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 +

27. Based on this information, can you make a prediction about the products of DNA from different sources cut with the same restriction enzymes?
Will the RFLP patterns produced by gel electrophoresis produced by DNA mapping be the same or different if you use just one restriction enzyme?
Do you have to use many restriction enzymes to find differences between individuals? Justify your prediction.
Can you make a prediction about the RFLP patterns of identical twins cut with the same restriction enzymes? How about the RFLP patterns of fraternal twins or triplets?
Now that you understand the basic idea of genetic mapping by using restriction enzymes, let’s explore how DNA fragments can be used to make a genetic profile.

28. • Why do DNA fragments migrate through the gel from the negatively charged pole to the positively charged pole?

29. Lesson 3 Electrophoresis of Your DNA Samples
Review Questions
1. The electrophoresis apparatus creates an electrical field with positive and negative poles at the ends of the gel. DNA molecules are negatively charged. To which electrode pole of the electrophoresis field would you expect DNA to migrate? (+ or -)? Explain.
2.What color represents the negative pole?
3. After DNA samples are loaded into the sample wells, they are “forced” to move through the gel matrix. What size fragments (large vs. small) would you expect to move toward the opposite end of the gel most quickly? Explain.
4. Which fragments (large vs. small) are expected to travel the shortest distance from the well? Explain.

30. DNA Schematic O O Phosphate Sugar Phosphate Sugar O O P O Base O CH2 OOH

31. DNA Fingerprinting Procedures Day Two

32. Components of an Electrophoresis System
Power supply and chamber, a source of negatively charged particles with a cathode and anode
Buffer, a fluid mixture of water and ions
Agarose gel, a porous material that DNA migrates through
Gel casting materials
DNA ladder, mixture of DNA fragments of known lengths
Loading dye, contains a dense material and allows visualization of DNA migration
DNA Stain, allows visualizations of DNA fragments after electrophoresis
Ions: atoms that have a positive or negative charge because they have lost or gained electrons.
Electrophoresis: migration of ions at different speeds is a basic principal

33. Agarose Electrophoresis Loading
• Electrical current carries negatively-charged DNA through gel towards positive (red) electrode
Buffer
Dyes
Agarose gel
Power Supply

34. Agarose Gel A porous material derived from red seaweed
Acts as a sieve for separating DNA fragments; smaller fragments travel faster than large fragments
Concentration affects DNA migration
Low conc. = larger pores better resolution of larger DNA fragments
High conc. = smaller pores better resolution of smaller DNA fragments

35. Electrophoresis Buffer
TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA) are the most common buffers for duplex DNA
Establish pH and provide ions to support conductivity
Concentration affects DNA migration
Use of water will produce no migraton
High buffer conc. could melt the agarose gel

36. Agarose Electrophoresis Running
• Agarose gel sieves DNA fragments according to size
– Small fragments move farther than large fragments
Gel running
Power Supply

37. PROCEDURE: follow procedure recommended by BioRad for this lab-

38. Allows DNA visualization after gel electrophoresis Ethidium Bromide
DNA Staining
Allows DNA visualization after gel electrophoresis
Ethidium Bromide
Bio-Safe DNA stains

39. Analysis of Stained Gel
Determine
restriction fragment
sizes
• Create standard curve using DNA marker
• Measure distance traveled by restriction fragments
• Determine size of DNA fragments
Identify the related
samples

41. What observations can you make?
What quantitative measurements can you make?
1. Examine the “ideal” or mock gel shown in Figure 5 that includes DNA samples that have been cut with three restriction enzymes, BamHI, EcoRI, and HindIII, to produce RFLPs (fragments). Sample D is DNA that has not been cut with enzyme(s).
DNA cut with HindIII provides a set of fragments of known size and serves as a standard for comparison.

43. 2. Using the ideal gel shown in Figure 5 (using lambda DNA), measure the distance (in cm:) that each fragment migrated from the origin (the well). (Hint: For consistency, measure from the front end of each well to the front edge of each band, i.e., the edge farthest from the well.). Enter the measured distances into Table 1. (See * and ** notes below the table for an explanation for why there are only six bands seen but more fragments.)

44. Lambda Phage DNA Genomic DNA of a bacterial virus
Attacks bacteria by inserting its nucleic acid into the host bacterial cell
Replicates rapidly inside host cells until the cells burst and release more phages
Harmless to man and other eukaryotic organisms

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

53. Extension activity- Plasmid mapping 10 pts. E.C.