Introduction to Gel Electrophorsis

Model of DNA

DNA is Comprised of Four Base Pairs

Deoxyribonucleic Acid

(DNA)

DNA Schematic

OCH2

O

P O

O

OBase

CH2

O

P

O

O

O

Base

OH

Sugar

Sugar

O

Phosphate

Phosphate

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

• 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

5 vs 3 Prime Overhang

• Generates 5 prime overhang

Enzyme cuts

Common Restriction Enzymes EcoRI

– Eschericha coli– 5 prime overhang

Pstl– Providencia stuartii– 3 prime overhang

The DNA DigestionReaction Restriction Buffer provides

optimal conditions

• NaCI provides the correct ionic strength

• Tris-HCI provides the proper pH

• Mg2+ is an enzyme co-factor

AgaroseElectrophoresis

Loading

• Electrical current carries negatively-charged DNA through gel towards positive (red) electrode

Power Supply

Buffer

Dyes

Agarose gel

AgaroseElectrophoresis

Running

• Agarose gel sieves DNA fragments according to size– Small fragments

move farther than

large fragments

Power Supply

Gel running

Analysis of Stained Gel

Determinerestriction fragmentsizes

• Create standard curve using DNA marker

• Measure distance traveled by restriction fragments

• Determine size of DNA fragments

Identify the relatedsamples

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 24.0100

1,000

10,000

100,000

0 5 10 15 20 25 30

Distance, mm

Siz

e, b

ase

pai

rsB

A

Fingerprinting Standard Curve: Semi-log

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