Transcript Introduction to Gel Electrophorsis

Introduction to Gel
Electrophorsis
Model of DNA
DNA is Comprised of
Four Base Pairs
Deoxyribonucleic Acid
(DNA)
O
Phosphate
O P O
O
CH2
Base
O
Sugar
DNA Schematic
O
Phosphate
O P O
Base
O
CH2
O
Sugar
OH
DNA Restriction
Enzymes
• Evolved by bacteria
to protect against
viral DNA infection
• Endonucleases =
cleave within DNA
strands
• Over 3,000 known
enzymes
Enzyme Site
Recognition
Restriction site
Palindrome
• Each enzyme digests
(cuts) DNA at a
specific sequence =
restriction site
• Enzymes recognize
4- or 6- base pair,
palindromic
sequences
(eg GAATTC)
Fragment 1
Fragment 2
Enzyme cuts
5 vs 3 Prime
Overhang
• Generates 5 prime
overhang
Common Restriction
Enzymes
EcoRI
– Eschericha coli
– 5 prime overhang
Pstl
– Providencia stuartii
– 3 prime overhang
The DNA Digestion
Reaction
Restriction Buffer provides
optimal conditions
• NaCI provides the correct
ionic strength
• Tris-HCI provides the proper
pH
• Mg is an enzyme co-factor
2+
Agarose
Electrophoresis
Loading
• Electrical
current
carries
negativelycharged
DNA
through gel
towards
positive
(red)
electrode
Buffer
Dyes
Agarose gel
Power Supply
Agarose
Electrophoresis
Running
• Agarose gel
sieves DNA
fragments
according to
size
– Small
fragments
move
farther than
large
fragments
Gel running
Power Supply
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
Identify the related
samples
Fingerprinting Standard Curve: Semi-log
Molecular Weight
Determination
Size (bp)
100,000
Distance (mm)
23,000 11.0
9,400 13.0
6,500 15.0
Size, base pairs
10,000
B
1,000
4,400 18.0
2,300 23.0
2,000 24.0
100
0
5
10
15
Distance, mm
20
A
25
30
Agarose Gel Electrophoresis
• The standard method for separating DNA
fragments is electrophoresis through agarose
gels.
Agarose Gel Electrophoresis
• The standard method for separating DNA
fragments is electrophoresis through agarose
gels.
• Agarose is a polysaccharide like agar or pectin
derived from seaweed
Agarose Gel Electrophoresis
• The standard method for separating DNA
fragments is electrophoresis through agarose
gels.
• Agarose is a polysaccharide like agar or pectin
derived from seaweed
• It dissolves in boiling water and then gels as it
cools
Agarose Gel Electrophoresis
• A comb is placed in the
liquid agarose after it
has been poured
• Removing the comb
from the hardened gel
produces a series of
wells used to load the
DNA
Agarose Gel Electrophoresis
• DNA is applied to a slab
of gelled agarose
Agarose Gel Electrophoresis
• DNA is applied to a slab
of gelled agarose
• The sample is loaded
with a loading buffer—
containing dyes and
glycerol or sugar
Agarose Gel Electrophoresis
• DNA is applied to a slab
of gelled agarose
• The sample is loaded
with a loading buffer—
containing dyes and
glycerol or sugar
• Electric current is
applied across the gel
Agarose Gel Electrophoresis
• DNA is negatively
charged (due to PO4)
Agarose Gel Electrophoresis
• DNA is negatively
charged (due to PO4)
• Migrates from the
negative (black)
electrode to the
positive (red) electrode.
Agarose Gel Electrophoresis
• Rate of migration of DNA through agarose
depends on the size of DNA
Agarose Gel Electrophoresis
• Rate of migration of DNA through agarose
depends on the size of DNA
• Smaller DNA fragments move more quickly
Agarose Gel Electrophoresis
• Rate of migration of DNA through agarose
depends on the size of DNA
• Smaller DNA fragments move more quickly
• Rate of migration is inversely proportional to
the log10 of molecular weight
Agarose Gel Electrophoresis
Agarose Gel Electrophoresis
• Concentration of agarose also affects
migration
Agarose Gel Electrophoresis
• Concentration of agarose also affects
migration
• Higher concentration of agarose, the more it
retards the movement of all DNA fragments
Agarose Gel Electrophoresis
• Concentration of agarose also affects
migration
• Higher concentration of agarose, the more it
retards the movement of all DNA fragments
• Small DNA fragments require higher
concentrations of agarose/ Lg fragments low
concentrations
Agarose Gel Electrophoresis
• Agarose gels must be prepared and run in a
buffer containing ions.
Agarose Gel Electrophoresis
• Agarose gels must be prepared and run in a
buffer containing ions.
• Ions are charged particles (like those found in
salt) and are necessary to carry a charge
Agarose Gel Electrophoresis
• During electrophoresis water undergoes
hydrolysis : H2O  H+ and OH-
Agarose Gel Electrophoresis
• During electrophoresis water undergoes
hydrolysis : H2O  H+ and OH• The anode (+ /red) pole becomes alkaline
because OH- will accumulate at this pole
• The cathode (-/black) pole becomes acidic
because H+ will accumulate at this pole
Agarose Gel Electrophoresis
• Buffers prevent the pH from changing by
reacting with the H+ or OH- products
Agarose Gel Electrophoresis
• The buffer is either TBE or TAE
– TBE is made with Tris/Boric Acid/EDTA
– TAE is made with Tris/Acetic Acid/ EDTA
Agarose Gel Electrophoresis
• The voltage applied to the gel affects how
quickly the gel runs
Agarose Gel Electrophoresis
• The voltage applied to the gel affects how
quickly the gel runs
• The higher the voltage, the more quickly the
gel runs………But that often reduces the
quality of the DNA separation
Agarose Gel Electrophoresis
• The voltage applied to the gel affects how
quickly the gel runs
• The higher the voltage, the more quickly the
gel runs………But that often reduces the
quality of the DNA separation
• >>>>>>>>>>It also generates heat which
reduces the quality of the DNA separation
Agarose Gel Electrophoresis
• To make DNA fragments visible after
electrophoresis, the DNA must be stained
Agarose Gel Electrophoresis
A gel stained with Methylene blue