1. Restriction Analysis of Plasmid DNA
Restriction Analysis Pitt Kit
9/11/2018
Restriction Analysis of Plasmid DNA

2. Restriction Enzymes

3. Each restriction enzyme cuts DNA wherever its recognition site appears.
Each restriction enzyme recognizes a particular sequence of nucleotides, called its restriction site. Many recognition sites are palindromes.
BamHI
…NNNGGATCCNNN… …NNNG GATCCNNN…
…NNNCCTAGGNNN… …NNNCCTAG GNNN…
HindIII
…NNNAAGCTTNNN… …NNNA AGCTTNNN…
…NNNTTCGAANNN… …NNNTTCGA ANNN…

4. A “restriction-modification” system.
If bacteria produce restriction enzymes, why doesn’t their own DNA get cut up?
Restriction Enzyme
Modification Enzyme
Cuts DNA anywhere the recognition sequence occurs. Will not cut if DNA is methylated (has –CH3 groups added)
Methylases act at same recognition site as restriction enzyme Protects bacteria’s own DNA from its own restriction enzymes Foreign DNA is not protected
A “restriction-modification” system.

5. Restriction Enzymes Create Either “Blunt” Ends or “Sticky” Ends with Overhangs
BamHI
5’…NNNGGATCCNNN…3’ 5’…NNNG GATCCNNN…3’
3’…NNNCCTAGGNNN…5’ 3’…NNNCCTAG GNNN…5’
blunt end
SmaI
5’…NNNCCCGGGNNN…3’ 5’…NNNCCC GGGNNN…3’
3’…NNNGGGCCCNNN…5’ 3’…NNNGGG CCCNNN…5’
3’ overhang
PstI
5’…NNNCTGCAGNNN…3’ 5’…NNNGCTGCA GNNN…3’
3’…NNNGACGTCNNN…5’ 3’…NNNG ACGTCNNN…5’

6. Ends Produced By The Same Enzyme Can Be Rejoined By Ligation
EcoRI
5’…CCCGAATTCCCC…3’ AATTCCCC…3’
3’…GGGCTTAAGGGG…5’ GGGG…5’
Base Pairs Re-Form
EcoRI
5’…AAAGAATTCAAA…3’ 5’…AAAG
3’…TTTCTTAAGTTT…5’ 3’…TTTCTTAA
After Ligation with DNA Ligase
5’…AAAGAATTCCCC…3’
3’…TTTCTTAAGGGG…5’

7. All blunt ends are cohesive.
Cohesive Ends Produced By Different Enzymes Can Be Rejoined By Ligation
BglII
5’…CCCAGATCTCCC…3’ GATCTCCC…3’
3’…GGGTCTAGAGGG…5’ AGGG…5’
These sticky ends share the same overhang sequence
BamHI
5’…AAAGGATCCAAA…3’ 5’…AAAG
3’…TTTCCTAGGTTT…5’ 3’…TTTCCTAG
After Ligation with DNA Ligase
5’…AAAGGATCTCCC…3’
3’…TTTCCTAGAGGG…5’
All blunt ends are cohesive.

8. Average Frequency of Recognition Sites Along a DNA Molecule
4-nucleotide recognition sequence
HaeIII GGCC
occurs once every 44 = 256 bp
6-nucleotide recognition sequence
EcoRI GAATTC
occurs once every 46 = 4,096bp
8-nucleotide recognition sequence
NotI GCGGCCGC
occurs once every 48 = 65,536bp

9. Using Restriction Enzymes to Genetically Engineer Recombinant DNAs
Non-Recombinant
Choose enzymes that yield cohesive ends
Cut plasmid DNA
Recombinant
Ligate with DNA Ligase
Cut insert DNA

10. Restriction Fragment Length Polymorphism (RFLP) Analysis
MstII recognizes the sequence CCTNAGG (“N” can be any nucleotide). The mutation that causes sickle- cell anemia eliminates a MstII recognition site.
Normal …ProGluGlu…
…CCTTAGG……………………………………………CCTGAGGAG………CCTTAGG…
Mutant …ProValGlu…
…CCTTAGG……………………………………………CCTGTGGAG………CCTTAGG…
1.2kb fragment
0.2kb fragment
1.4kb fragment

11. Session 1: Restriction Digest Reactions

12. Your sample is EITHER plasmid DNA pAMP or pKAN. Which do you have?
How could you tell them apart?
What’s different between them?

13. Each restriction enzyme cuts DNA wherever its recognition site appears.
Each restriction enzyme recognizes a particular sequence of nucleotides, called its restriction site. Many recognition sites are palindromes.
BamHI
…NNNGGATCCNNN… …NNNG GATCCNNN…
…NNNCCTAGGNNN… …NNNCCTAG GNNN…
HindIII
…NNNAAGCTTNNN… …NNNA AGCTTNNN…
…NNNTTCGAANNN… …NNNTTCGA ANNN…

14. A restriction map identifies where restriction sites appear along a DNA.
BamHI cuts here
HindIII cuts here
What will be different between the DNA fragments produced by cutting pAMP vs. pKAN with BamHI & HindIII?

15. DNAs can be distinguished from each other by restriction mapping.
3755 bp
2332 bp
784 bp
1875 bp
1904 – 1120 = 784
4539 – 784 = 3755

16. Glove Up! Put on a pair of lab gloves S, M, L, XL available
Most hands will fit in M or L gloves.
Try those sizes first unless you have particularly small or large hands.
Made of nitrile (no latex = no allergies)

17. Micropipetting Review
1st Step, 1st Stop.
2nd Step, 2nd Stop.
Micropipetting Review
FIRST STEP: Measuring
SECOND STEP: Dispensing
Choose a micropipette whose range spans the desired volume. Set the micropipette to the desired volume by turning the plunger knob. Don’t force past the pipette’s limits, as this breaks the pipette! Place a tip on the micropipette, matching tip and plunger colors. Depress plunger to the FIRST STOP and HOLD. Place the tip into the liquid. Slowly release the plunger, keeping tip in liquid.
Place tip against side of tube, near bottom. Depress plunger to the SECOND STOP and HOLD. Remove tip from tube while holding down plunger. Release plunger. Throw away used tip using the ejector button. Use a new tip each time you pipet.

18. Label a Restriction Digest Tube
From the jar with the white screw cap, remove one 1.5ml microtube. With a lab marker, label the lid of the microtube with your period number and the first initials of each team member. P1
TDH

19. Add Plasmid DNA
Your team was given a sample of either pAMP or pKAN plasmid DNA in a tube labeled “DNA” and a number between 1 and 12.
From this tube, use your micropipette to measure 5μl (microliters) of plasmid DNA and transfer it to your Restriction Digest tube.
At 0.1μg/μl,
this 5μl contains
0.5μg or 500ng of DNA.
DNA 1…12
5μl P1
TDH

20. Add Water
From the tube labeled H2O, measure 9μl of water and transfer it to your Restriction Digest tube.
H2O
9μl P1
TDH

21. Add Restriction Reaction Buffer
Enzymes require a chemical environment of the right pH and concentration of ions. The 5X restriction buffer is a concentrated mix that provides the environment needed for the restriction enzymes to work properly.
From the tube labeled 5X RE Buffer, measure 4μl of 5x Restriction Digest Buffer and transfer it to your Restriction Digest tube.
5X RE Buffer
4μl P1
TDH

22. Add Restriction Enzymes
You will cut your plasmid DNA with two restriction enzymes: BamHI and HindIII.
From the tube labeled BamHI + HindIII measure 2μl of the BamHI and HindIII mix and transfer it to your Restriction Digest tube.
BamHI + HindIII
2μl P1
TDH

23. Incubate the Restriction Digest Reaction
Close the cap on your Restriction Digest tube and place it in the heating block set at 37°C. The restriction enzymes work best at 37°C. The reactions will incubate for one hour, then be stored in a freezer until you examine them using gel electrophoresis.

24. Session 2: Gel Electrophoresis

25. Prepare Your Samples for Loading
Restriction Analysis Pitt Kit
9/11/2018
Prepare Your Samples for Loading
Add 4µl of the 6X Loading Dye to your restriction digest sample.
If your liquids are sticking separately to the side of the tube, flick the tube with your finger and tap the bottom gently on your lab bench, or spin briefly in microcentrifuge to collect entire sample at bottom of tube.

26. Load Your Sample On The FlashGel
When called, bring to the FlashGel:
Your DNA sample
Micropipette with tip
Load 6μl of your sample into a well.

27. Restriction Analysis Pitt Kit
9/11/2018
Write your team initials or team number below the well into which you loaded your sample.
Lane 1 2 3 4 5 6 7 8 9 10 11 12 13
1kb ladder
Period ____
Teacher: Print out copies of this slide to place near each gel each period. Have students write their initials or team number in the lane into which their sample was loaded.

28. Run the Gel
A power supply provides current to the electrodes and through the buffer and gel. The progress of migration through the gel is monitored with tracking dyes that are visible without the transilluminator. 1.2% Flash Gel 200 V 8 minutes

29. Analysis of Gel Results

30. Restriction Mapping Can Be Used To Identify Unknown DNAs
3755 bp
2332 bp
784 bp
1875 bp

31. Restriction Analysis Pitt Kit
9/11/2018 1 2 3 4 5 6 7 8 9 10 11 12 13
Restriction Fragment Sizes
pAMP:
3755,
784
pKAN:
2332
1875
Promega BenchTop 1kb Ladder
Period #1
Teacher: After capturing a digital image with the Flash Gel software, return to this slide. Right-click on the gel image, choose “Change Picture…”, and browse to the gel image you just captured. That will replace this image with your new one.
1.2% 200V 8min

32. What Questions Do YOU Have?