restriction mapping of a bacterial plasmid (danna and nathans, 1971)
TRANSCRIPT
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
Size
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