restriction digestion and gel electrophoresis laboratory
TRANSCRIPT
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Restriction Digestion and Gel Electrophoresis
Laboratory
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DNA Restriction Enzymes
• Evolved by bacteria to protect against viral DNA infection
• Endonucleases = cleave within DNA strands
• Over 3,000 known enzymes
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Enzyme Site Recognition
• Each enzyme digests (cuts) DNA at a specific sequence = restriction site
• Enzymes recognize 4- or 6- base pair, palindromic sequences (eg GAATTC)
Palindrome
Restriction site
Fragment 1 Fragment 2
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5 vs 3 Prime Overhang
Generates 5 prime overhang Enzyme cuts
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Common Restriction Enzymes
EcoRI– Eschericha coli– 5 prime overhang
Pstl– Providencia stuartii– 3 prime overhang
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The DNA Digestion Reaction
• Restriction Buffer provides optimal conditions– NaCI provides the correct ionic strength– Tris-HCI provides the proper pH– Mg2+ is an enzyme co-factor
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DNA DigestionTemperature
Why incubate at 37°C?– Body temperature is optimal for these and
most other enzymes
What happens if the temperature is too hot or cool? – Too hot = enzyme may be denatured – Too cool = enzyme activity lowered,
requiring longer digestion time
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Restriction Fragment Length PolymorphismRFLP
Allele 1
Allele 2
GAATTCGTTAAC
GAATTCGTTAAC
CTGCAGGAGCTC
CGGCAGGCGCTC
PstI EcoRI
1 2 3
3Fragment 1+2Different Base PairsNo restriction site
M A-1 A-2
Electrophoresis of restriction fragments
M: MarkerA-1: Allele 1 FragmentsA-2: Allele 2 Fragments
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Agarose Electrophoresis Loading
• Electrical current carries negatively-charged DNA through gel towards positive (red) electrode
Power Supply
Buffer
Dyes
Agarose gel
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AgaroseElectrophoresis
Running • Agarose gel sieves DNA
fragments according to size
–Small fragments move farther than large fragments
Power Supply
Gel running
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Analysis of Stained Gel
Determine restriction fragment sizes
–Create standard curve using DNA marker
–Measure distance traveled by restriction fragments
–Determine size of DNA fragments
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• Can be used to determine relative size of a DNA fragment (e.g. size of a gene)
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Molecular Weight Determination
Size (bp) Distance (mm)
23,000 11.0 9,400 13.0
6,500 15.0
4,400 18.0
2,300 23.0
2,000 24.0
100
1,000
10,000
100,000
0 5 10 15 20 25 30
Distance, mm
Siz
e, b
ase
pai
rs
B
A
Fingerprinting Standard Curve: Semi-log
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Equipment
Micropipet—measures small volumes of liquid (l –ml)
1000 l = 1 ml
Microfuge tubes
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Minicentrifuge—pools liquid, solids to bottom of centrifuge tube
Samples of balanced rotor configurations
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Agarose gel--porous