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

Restriction Mappingof 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

Plasmids

Plasmids

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)

EcoRI 5’ G/AATTC 3’3’ CTTAA/G 5’

AATTCGTGCGATGCAT GCACGCTACGTACGTAGCGTAGCGGCATCGCATCGCTTAA

EcoRI 5’ G/AATTC 3’3’ CTTAA/G 5’

AATTCGTGCGATGCAT GCACGCTACGTA

CGTAGCGTAGCGGCATCGCATCGCTTAA

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’

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’

Restriction Enzyme Examples

4 cutter

6 cutters

8 cutter

Restriction Map

Restriction Digest

EcoRI 4361 bp

HindIII 4361 bp

BamHI 4361 bp

AccI 1593 bp 2768 bp

ApaLI 2617 bp 1246 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

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Gel Visualized Under UV Light

Plasmids on Agarose Gels

uncut cut 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 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

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 water1ul enzyme

Incubate for 1 hour at 37C

Add 4ul “stop mix” (50% glycerol, 1% SDS, 50mM EDTA, 0.1% bromphenyl blue)

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’

Restriction Enzymes for This Experiment

Your Gel Today

Size standards

Bam

HI

EcoR

I

HindIII

PstI

ScaI

XbaI

XhoI

Size standards

Your Gel Today

Size

Bam

HI

EcoR

I

HindIII

PstI

ScaI

XbaI

XhoI

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