basic principles and applications of electrophoresis

電泳

Theory of ElectrophoresisThe movement of a charged molecule subjected to an electric field is represented by the following equation:

V = Eqf

V: the velocity of the molecule

E: the electric field in volts/cm

q: the net charge on the molecule

f: frictional coefficient, which depend on the mass and shape of the molecule

Gel Electrophoresis of DNA

Agarose slab gel submerged in buffer

Wells for sample loading

Cathode (-)

Direction for DNA migration Anode (+)

Agarose is a polysaccharide derived from seaweed, which forms a solid gel when dissolved in aqueous solution. When an electric field is applied to an agarose gel in the presence of a buffer solution which will conduct electricity, DNA fragments move through the gel towards the positive electrode (DNA is highly negatively charged) at a rate which is dependent on its size and shape.

Gel Electrophoresis of DNA

For linear DNA molecules, they have uniform shape and charge to mass ratio. The electrophoretic mobility of the DNA molecule is influenced primarily by the molecular size: The larger molecules are retarded by the molecular sieving effect of the gel, and the small molecules have greater mobility.

Gel Electrophoresis of DNA

• The DNA can be stained by the inclusion of ethidium bromide in the gel, or by soaking the gel in a solution of ethidium bromide after electrophoresis. The DNA shows up as an orange band on illumination by UV light. Alternatively, methylene blue can be used to stain DNA.

• Gels composed of polyacrylamide can separate DNA molecules that differ in length by only one nucleotide and are used to determine the base sequence of DNA. Agarose gels are used to separate DNA fragments that have larger size differences.

Digestion of DNA by Restriction Enzymes

Before the electrophoresis, DNA is digested by restriction enzymes into small fragments of DNA.

GAATTCCTTAAG

G AATTCCTTAA G

EcoRI

Restriction enzymes - enzymes isolated from bacteria that cut DNA at specific sites(restriction sites)EcoRI - 5'- G A A T T C -3‘

3'- C T T A A G -5'

Properties of DNA-Double helixBuilding block(dA, dC, dG and dT)negatively charged at neutral pHAT and GC complementary pairing

Baterial plasmid DNAPlasmids are molecules of DNA that are found in bacteria separate from the bacterial chromosome.They: are small (a few thousand base pairs)usually carry only one or a few genes are circularhave a single origin of replication

+

BglI 2166

BglI 472

EcoRI 701

Plasmid DNA for digestion – pBluescript II SK+

Agarose

A linear polymer extracted from seaweed

Migration of DNA in agarose dependent on four factors

- molecular size of the DNA- agarose concentration- conformation of the DNA- applied current

wells

Anode(+)

Cathode(-)

1.5% agarosegel stained with methylene blue

DNA fragments of different sizes

Wells

Bromophenol blue

Xylene cyanol FF

Tracking dye

Methylene blue staining» to visualize the DNA fragments, stain agarose gel overnight with 1X methylene blue staining solution» safe alternative for DNA staining» easy available» non-carcinogenic

DNA fragments

Base pairs distance migrated(mm)

A B C D M

316 47

656 38

1808 231634 25

DNA fragments of different sizes

DNA fragments of known sizes