1. Restriction Enzymes and Digestion

2. What can you do with the Restriction Digestion and Analysis of Lambda DNA Kit?
Understand the use of restriction enzymes as biotechnology tools and the mechanics of a restriction enzyme digest
Become familiar with principals and techniques of agarose gel electrophoresis
Estimate DNA fragment sizes from agarose gel data
Understand the importance of restriction enzymes and their applications

3. What are restriction enzymes?
Evolved by bacteria to protect against viral DNA infection
1950s: discovery of primitive immune system in bacteria
1962: proof that in bacteria, an enzyme system recognized and destroyed foreign DNA while protecting its own
1960s: E.coli extracts isolated which cleaved phage DNA, but not useful
1970s: H. influenzae, HindII, extract isolated with no modification activity and cleavage within the restriction site
Endonucleases = cleave within DNA strands
Exonucleases = digest from the ends of DNA molecules
Types I, II, and III
3,139 known enzymes

4. How does it work? Enzyme Site Recognition
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

5. Palindromic Sequences
Palindrome: ‘reads’ the same both ‘forward’ and ‘backward’. Enzymes ‘cut’ DNA in non-coding regions. Individuals (except ID twins) have various #s of sequences that repeat over and over (called STRs) in these regions, which is why DNA fingerprints are over 99.9% accurate.
Enzyme cuts 
5’ GAATTC 3’
3’ CTTAAG 5’
5’ G ’
3’ CTTAA 5’
5’ AATTC 3’
3’ G 5’
Sticky ends- have ‘overhang’
Blunt ends: no ‘overhang’ (not as ‘specific’ in the direction it is ‘read’)
5’ GAA TTC 3’
3’ CTT AAG 5’

6. Common Restriction Enzymes
5’ GAATTC 3’
3’ CTTAAG 5’
EcoRI
Escherichia coli
Cuts between G and A
HindIII
Haemophilus influenzae
Between A and A
1st free-living organism to have its entire chromosome sequenced
Type b, Hib, was the leading cause of bacterial meningitis among children under 5 years old in the US, before development of a vaccine.
*Do NOT have to memorize where specific enzymes ‘cut’!
5’ AAGCTT 3’
3’ TTCGAA 5’

7. What is needed for restriction digestion?
Template DNA, uncut DNA, often bacterial phage DNA
Restriction enzyme(s), to cut template DNA
Restriction Buffer, to provide optimal conditions for digestion
Water Bath

8. Restriction Enzyme Digestion
Restriction Buffer provides optimal conditions:
NaCl provides correct ionic strength
Tris-HCl provides the proper pH
Mg2+ is an enzyme co-factor
Different enzymes have different optimal buffers; Manufacturers package enzymes with buffers for ease of use

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

10. How do we visualize the DNA?
Agarose Gel Electrophoresis

11. Agarose Gel Electrophoresis
Electrolysis: the splitting of water using electricity
current splits water into hydrogen ions (H+) and hydroxyl ions (OH-)
Electrophoresis: a method of separating charged molecules in an electrical field; DNA has an overall negative charge
Used to separate DNA fragments by size

12. 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

13. - + Cathode Anode Buffer Dyes Agarose gel Power Supply
During electrophoresis, water is electrolyzed which generates protons (H+ ions)at the anode (positive) and hydroxyl ions (OH -1)at the cathode (negative). The cathode (negative) end of the electrophoresis chamber then becomes basic and the anode (positive) end becomes acidic.
The electrode at which electrons enter the gel box from the power supply (along the black wire) is called the cathode and is negative (-). The electrode at which electrons leave the box and re-enter the power supply (along the red wire) is called the anode and carries a positive charge (+). The flow of electrons sets up a potential energy difference between the electrodes. This is known as potential, and is measured in volts. It establishes an electric field through which the ions in the gel box fluid migrate. The migration of ions in the fluid creates electrical current which is measured in milliamperes (milliamps).

14. 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
A buffer is a chemical system that maintains a relatively constant pH even when strong acids or bases are added. Buffer solutions contain either a weak acid or weak base and one of their salts. Because a change in pH can alter the charge on a particle, it is important to use a buffer solution when separating during electrophoresis.

15. Agarose Gel A porous material derived from red seaweed
Acts as a sieve for separating DNA fragments; smaller fragments travel faster than large fragments
Plinko Model
Concentration affects DNA migration
Low conc. = larger pores better resolution of larger DNA fragments
High conc. = smaller pores better resolution of smaller DNA fragments
1% agarose
2% agarose

16. Loading Dye
DNA samples are loaded into a gel AFTER the tank has been filled with buffer, covering the gel
Contains a dense substance, such as glycerol, to allow the sample to "fall" into the sample wells
Contains one or two tracking dyes, which migrate in the gel and allow monitoring of how far the electrophoresis has proceeded.

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

18. Complete a Gel Electrophoresis simulation at:
Agarose Gel
DNA Fragments
Complete a Gel Electrophoresis simulation at:

19. Restriction Enzyme Digest and Analysis Procedures

20. Actual Results of Restriction Enzyme Digestion
Lane 1, DNA markers (HindIII lambda digest)
lane 2, uncut lambda DNA
lane 3, lambda DNA digested with PstI
lane 4, lambda DNA digested with EcoRI
lane 5, lambda DNA digested with HindIII

21. Analysis of DNA Fragments
Determine restriction fragment sizes
Create standard curve using DNA marker
Measure distance traveled by restriction fragments
Determine size of DNA fragments

22. DNA Marker Standard Curve
Size (bp) Distance (mm)
23, 9, 6, 4, 2, 2,

23. Factors Affecting Restriction Enzyme Digestion
Temperature, restriction enzymes are sensitive to prolonged periods of exposure to heat
Cross contamination of restriction enzymes
Buffer, optimum pH
Incubation temperature, maintain optimum temperature during restriction enzyme activity
And Finally…Don’t forget to ADD your restriction enzyme to the reaction!!!

24. Crime Scene Investigation
VNTR’s: number of repeats varies from 4 to 40 in different individuals
Variants inherited from parents; unrelated individuals not likely to have same repeats
Length of DNA depends on the number of repeats at each locus
Same 3 VNTR loci are analyzed for 3 individuals = 6 bands for each
Results can serve as a DNA fingerprint to exclude suspects