Restriction Mapping of a Bacterial Plasmid (Danna and Nathans, 1971)

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

Plasmids

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.

A Restriction Enzyme (BgII)

EcoRI5’ G/AATTC 3’ 3’ CTTAA/G 5’ AATTCGTGCGATGCAT GCACGCTACGTA CGTAGCGTAGCG GCATCGCATCGCTTAA

EcoRI5’ G/AATTC 3’ 3’ CTTAA/G 5’ AATTCGTGCGATGCAT GCACGCTACGTA CGTAGCGTAGCG GCATCGCATCGCTTAA

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

MseI 5’ A/T A A 3’ 3’ T A T/A 5’ BamHI5’ G/G A T C C 3’ 3’ C C T A G/G 5’ EcoRI5’ 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’ Restriction Enzyme Examples 4 cutter 6 cutters 8 cutter

Restriction Map

Restriction Digest EcoRI 4361 bp HindIII 4361 bp BamHI 4361 bp AccI 1593 bp2768 bp ApaLI 2617 bp1246 bp 498 bp

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

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

Electrophoresis power supply

Gel Visualized Under UV Light

Plasmids on Agarose Gels uncutcut once

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.

Today’s Experiment Restriction Digest of Plasmid Each lab pair you will be given a 300 µ l aliquot of plasmid DNA at a concentration of approximately 100 µ g/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

Restriction Digest of Plasmid For each restriction digest, mix: 5ul DNA = 0.5 ug DNA) 3ul 5x buffer (100mM NaCl, 10mM Tris-Hcl pH 7.5, 10mM MgCl 2, 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)

BamHI5’ G/G A T C C 3’ 3’ C C T A G/G 5’ EcoRI5’ 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’ Restriction Enzymes for This Experiment

Your Gel Today Size standardsBamHIEcoRIHindIIIPstIScaIXbaIXhoISize standards

Your Gel Today Size BamHI EcoRI HindIII PstI ScaI XbaIXhoISize