Student Principles Of Gel Electrophoresis

Principles of Gel Electrophoresis

• A day without electrophoresis is very rare in molecular biology labs, because this technique is the standard method used for analyzing, identifying and purifying fragments of DNA. It is also used for separating and analyzing RNAs and oligonucleotides. • Electrophoresis is a technique used to separate and purify macromolecules especially proteins and nucleic acids - that differ in size, charge or conformation.

• When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge. • Proteins have either a net positive or net negative charge, nucleic acids have a consistent negative charge (phosphate backbone) and migrate toward the anode.

• Proteins and nucleic acids are electrophoresed within a matrix or "gel" in the shape of a thin slab, with wells for loading the sample. • The gel is immersed within an electrophoresis buffer that provides ions to carry a current and some type of buffer to maintain the pH at a relatively constant value. ( Movie )

The gel itself is composed of either agarose or polyacrylamide, each of which have attributes suitable to particular tasks: • Agarose is a polysaccharide 0.5 to 2%w/v. Simply mix agarose powder with buffer solution, melt it by heating, and pour the gel ( physical process) . It is non-toxic. It is a relatively smooth gel. A large range but relatively low resolving power of separation. Varying agarose concentrations separate DNA fragments from about 100 to 50,000 bp.

Agarose (%)

Range of separation of linear DNA (in kilobases)

0.3

60 - 5

0.6

20 - 1

0.7

10 - 0.8

0.9

7 - 0.5

1.2

6 - 0.4

1.5

4 - 0.2

2.0

3 - 0.1

• Polyacrylamide is a cross-linked polymer of acrylamide (typically between 3.5 and 20% acrylamide) (chemical between acrylamide and bis-acrylamide) . Annoying to prepare where oxygen inhibits the polymerization therefore poured between glass plates (or cylinders). A hard gel and easy to recover in pure state.

Acrylamide powder itself is a potent neurotoxin. A rather small range but very high resolving power separation, separate molecules that differ in size by as little as 2% of their molecular weight . Separating DNA fragments of less than about 100 bp. DNA fragments differing is length by a single base pair are easily resolved. (Therfore ,for proteins, used extensively for separating and characterizing).

Percent Acrylamide ((19:1 bis-acrylamide

Size Range (# of basepairs)

20

6-100

15

25-150

12

40-200

8

60-400

5

80-500

3.5

1000-2000

• • • • • • •

The equipment and supplies necessary for conducting agarose gel electrophoresis (or PAGE)are relatively simple and include: An electrophoresis chamber and power supply Gel casting trays Sample combs to form sample wells in the gel Electrophoresis buffer, as Tris-acetate-EDTA Loading buffer and one or two tracking dyes e.g, Bromophenol blue and xylene cyanol (for monitoring) Ethidium bromide, a fluorescent dye used for staining An ultraviolet lightbox

3. Preparation of the gel 4. Microwave oven, casting tray, ethidium bromide (or soaking after electrophoresis), sample combs, cooling 5. Assembling, loading, and power on. 6. Migration and visualization (ultraviolet lightbox).

Figure 1

Optimizing factors in gel separation: a. Agarose (or polyacrylamide) concentration: Higher concentrations of agarose (or polyacrylamide) facilite separation of small DNAs

Figure 2 using same gel tray, .same voltage, same running time

b. Voltage: As the voltage is increased larger fragments migrate proportionally faster than small fragments. c. Electrophoresis buffer: Buffers not only establish a pH, but provide ions to support conductivity. DNA fragments will migrate at somewhat different rates in two buffers. But concentrated buffer could generate heat that causes gel to melt.

The rate of migration is determined by (1) the charge on the macromolecule, (2) the strength of the electrical field, (3) the size of the macromolecule, (4) the shape of the macromolecule, (5) the state of hydration of the macromolecule, (6) the viscosity of the solution, and (7) the pore size of the gel.

where v = the rate (velocity) of migration, E is the strength of the electrical field, z is the charge on the molecule and f is the frictional force on the molecule.

where η is the viscosity of the medium and r is the stokes radius of the molecule.

The gel serves two purposes. It serves to diffuse convective currents and to creates a molecular sieve (enhancing the separation based on molecular weight). Note: since nucleic acids are negatively charged urea –and not EDTA- is used to denature the DNA or RNA within the gel.

Mobility -distance from the well- is inversely proportional to the log10 of their molecular weight or number of base pairs (straight line).

Figure 3

DNA can be isolated from Agarose gel by different methods e.g Electroelution : Cut out the desired piece into dialysis tubing with a small amount of fresh electrophoresis buffer. Current will cause the DNA to migrate out of the agarose. Then DNA is precipated out by ethanol. DNA is isolated from polyacrylamide gel by cutting out the desired piece into elution buffer, incubation then spinning down the gel. Then DNA is precipated out by ethanol.

Southern blotting • Double-stranded DNA on the gel can be separated into single strands by denaturation in alkaline solution. • DNA is then (or at the same time) moved from the gel onto membrane (nitrocellulose or nylon) by pressuring the membrane and gel with a stack of paper towels. DNA will stick and permanently crosslink to the membrane by special treatment. • The membrane is then treated with a hybridization probe which is an isolated DNA (or RNA) molecule with a specific sequence that pairs with the sequence we are looking for.

• The probe DNA has to be labelled so that it can be detected, usually by incorporating radioactivity or tagging the molecule with a fluorescent or chromogenic dye. • Excess probe is washed from the membrane, and the pattern of hybridization is visualized on x-ray film by autoradiography.

Figure 4 Blotting

(Figure 5 e-d steps) DNA is extracted from the cells and purified. A large piece of DNA is chopped into smaller pieces using a restriction enzyme. The DNA is loaded into a well #2of the gel matrix. Well #1 contains standards of known DNA fragments to quantitate the size.Well #3 contains unrestricted (intact) DNA .

Figure 6 A-B

Northern blotting A technique used to study gene expression. Similar to southern blotting with the key difference that RNA, rather than DNA, is the substance being analyzed by electrophoresis and detection with a hybridization probe. Formaldehyde is used to denature DNA on gel , because the sodium hydroxide treatment used in the southern blot procedure would degrade the RNA. The hybridization probe may be made from DNA or RNA. A variant of the procedure known as the Reverse Northern Blotting : a collection of isolated DNA fragments is affixed to the membrane and the probe is RNA extracted from a tissue and radioactively labelled (DNA microarrays).

Applications DNA Fingerprinting. e.g.the identification of particular DNA molecules by the band patterns they yield in gel electrophoresis after being cut with various restriction enzymes (Viral DNA, plasmid DNA, and particular segments of chromosomal DNA can all be identified). DNA Recombinant Technology. e.g. the isolation and purification of individual fragments containing interesting genes, which can be recovered from the gel with full biological activity … The Human Genome Project. e.g. to map the entire human genome and to determine the genetic difference and the evolutionary relationship among species of plants and animals.