Restriction Mapping of a Bacterial Plasmid

1. Restriction Mappingof a Bacterial Plasmid (Danna and Nathans, 1971)

2. Plasmids • Small, autonomously replicating extrachromosomal pieces of DNA found in bacteria, archaea and some eukaryotes • Usually circular • Contain an origin of replication • Usually contain genes conferring advantage on host (e.g. antibiotic resistance) • Play an important role in conjugation (bacterial sex) and lateral gene transfer

3. Plasmids

4. Plasmids

5. Plasmids

6. Plasmids

7. Restriction Enzymes Restriction endonucleases are bacterial enzymes that cleave double-stranded DNA at specific sequences (usually 4-8 basepairs in length) Discovered in 1970 by Tom Kelly and Ham Smith.

8. A Restriction Enzyme (BgII)

9. EcoRI 5’ G/AATTC 3’ 3’ CTTAA/G 5’ AATTCGTGCGATGCAT GCACGCTACGTA CGTAGCGTAGCG GCATCGCATCGCTTAA

10. EcoRI 5’ G/AATTC 3’ 3’ CTTAA/G 5’ CGTAGCGTAGCG GCATCGCATCGCTTAA AATTCGTGCGATGCAT GCACGCTACGTA

11. Restriction Enzymes • > 3,500 different restriction enzymes • > 270 different specificities • Named for species and strain from which they were originally isolated: • Escherichia coli R  EcoRI • Bacillus amyloliquefaciens H  BamHI • Providencia stuartii  PstI

12. Restriction Enzyme Examples 4 cutter MseI 5’ A/T A A 3’ 3’ T A T/A 5’ BamHI 5’ G/G A T C C 3’ 3’ C C T A G/G 5’ EcoRI 5’ G/A A T T C 3’ 3’ C T T A A/G 5’ HindIII 5’ A/A G C T T 3’ 3’ T T C G A/A 5’ NotI 5’ G C/G G C C G C 3’ 3’ C G C C G G/C G 5’ 6 cutters 8 cutter

13. Restriction Map

14. 1593 bp 2768 bp 2617 bp 1246 bp 498 bp Restriction Digest EcoRI 4361 bp HindIII 4361 bp BamHI 4361 bp AccI ApaLI

15. Agarose Gels • To visualize the results of a restriction digest, you need to separate the different fragments of DNA, and determine their size • We will do this by agarose gel electophoresis

16. Agarose • Agarose is very water soluble polysaccharide • Forms porous, aqueous gels after heating and cooling

17. 6000 4000 3000 2000 1500 1000 500 200 Electrophoresis - power supply +

18. Gel Visualized Under UV Light

19. Plasmids on Agarose Gels uncut cut once

20. EXPERIMENT 1: MAPPING DNA • Session 1: single enzyme digests and agarose gel #1 • Session 2: double digests and agarose gel #2 • Session 3: more digests and agarose gel #3 • Session 4: run and blot gel #4 • Session 5: complete DNA blot.

21. Today’s ExperimentRestriction Digest of Plasmid Each lab pair you will be given a 300µl aliquot of plasmid DNA at a concentration of approximately 100µg/ml in: TE:10mM Tris-HCl, 1mM EDTA pH 8 NOTE: This is a stock solution, you will only use a small amount for each reaction

22. Restriction Digest of Plasmid For each restriction digest, mix: 5ul DNA (@100ug/ul = 0.5 ug DNA) 3ul 5x buffer (100mM NaCl, 10mM Tris-Hcl pH 7.5, 10mM MgCl2, 50 ug/ul) 6ul sterile water 1ul enzyme Incubate for 1 hour at 37C Add 4ul “stop mix” (50% glycerol, 1% SDS, 50mM EDTA, 0.1% bromphenyl blue)

23. Restriction Enzymes for This Experiment BamHI 5’ G/G A T C C 3’ 3’ C C T A G/G 5’ EcoRI 5’ G/A A T T C 3’ 3’ C T T A A/G 5’ HindIII 5’ A/A G C T T 3’ 3’ T T C G A/A 5’ PstI 5’ C T G C A/G 3’ 3’ G/A C G T C 5’ ScaI 5’ A G T/A C T 3’ 3’ T C A/T G A 5’ XbaI 5’ T/C T A G A 3’ 3’ A G A T C/T 5’ XhoI 5’ C/T C G A G 3’ 3’ G A G C T/C 5’

24. Your Gel Today Size standards Size standards BamHI EcoRI HindIII PstI ScaI XbaI XhoI

25. 6000 6000 4000 4000 3000 3000 2000 2000 1500 1500 1000 1000 500 500 200 200 Your Gel Today Size Size BamHI HindIII EcoRI XbaI XhoI ScaI PstI