Gel Electorphoresis

TEKNIK ANALISIS ASAM NUKLEAT (DNA)

OH

Phosphate

HO

H

+

Nucleotide

P

O

Base N

H O 5’CH2 4’

NH2

H N

O

Sugar

3’

2’

OH

H OH

1’

N N Nucleoside

5’Phosphate group NH2

O N

O

P O

N

O

O HO

P

O

N

OH

H

H2O

N

O

NH2

N

CH2

O

CH2

O

O N

O CH2

N O

HN

O

N H2

N

H

N

CH2 O P HO

H

O

OH

H2O

N

5’Phosphate group HO

P

NH2

HO

P

O

H

O

H

E

3’Hydroxyl group

NH

O

2

N

N

CH2

H

O

O

N

O

N

H

O

HO

HN

OH

O

HO

H

N

N

O

3’Hydroxyl group

O

CH2

O

3

O

P

CH

S B A S E

ON PHATE BACKB SUGAR-PHOS HO

OH

Gel Electrophoresis  Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins  Separates DNA (or RNA or Protein) fragments on the basis of charge and size  DNA is an acid, it looses protons in basic buffers, thus it has a negative charge  By placing DNA in a gel, then applying a voltage accross the gel, the negatively charged DNA will move toward the positive pole  Large fragments lag behind while small fragments move throught the gel relatively rapidly  Agarose (a polysaccharide) or other gel matrices are difficult for large DNA fragments to move through

• DNA is negatively charged • When placed in an electrical field, DNA will migrate toward the positive pole (anode) • An agarose gel is used to slow the movement of DNA and separate by size

H 

O2  DNA

-

Power

+

• Polymerized agarose is porous, allowing for the movement of DNA Scanning Electron Micrograph of Agarose Gel (1×1 µm)



How fast will the DNA migrate? -Gel electrophoresis separates DNA according to size of the DNA -Small DNA move faster than large DNA

DNA small

-

Power

large

+

Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.

Estimation of the size of DNA molecules following restriction enzyme digestion, e.g. in restriction mapping of cloned DNA.  Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting  Separation of restricted genomic DNA prior to Southern transfer, or of RNA prior to Northern transfer. 

The advantages are that the gel is easily poured, does not denature the samples, and is physically firmer than polyacrylamide. The samples can also be recovered.  The disadvantages are that gels can melt during electrophoresis, the buffer can become exhausted, and different forms of genetic material may run in unpredictable forms. 







The most important factor is the length of the DNA molecule, smaller molecules travel farther. But conformation of the DNA molecule is also a factor. Increasing the agarose concentration of a gel reduces the migration speed and enables separation of smaller DNA molecules. The higher the voltage, the faster the DNA migrates. But voltage is limited by the fact that it heats and ultimately causes the gel to melt. High voltages also decrease the resolution (above about 5 to 8 V/cm). Conformations of a DNA plasmid that has not been cut with a restriction enzyme will move with different speeds (slowest to fastest): nicked or open circular, linearised, or supercoiled plasmid.





The most common dye used for agarose gel electrophoresis is ethidium bromide, usually abbreviated as EtBr. It fluoresces under UV light when intercalated into DNA (or RNA). Loading buffers are added with the DNA in order to visualize it and sediment it in the gel well. Negatively charged indicators keep track of the position of the DNA. Xylene cyanol and Bromophenol blue are typically used. They run at about 5000 bp and 300 bp respectively, but the precise position varies with percentage of the gel. Other less frequently used progress markers are Cresol Red and Orange G which run at about 125 bp and 50 bp.



 

Gel electrophoresis can be used for the separation of DNA fragments of 50 base pairs up to several megabases (millions of bases). However, it is normally used in a range of 100 bp to 20 kbp. Typical run times are about an hour. Small nucleic acids are better separated by polyacrylamide gels, In general higher concentrations of agarose are better for larger molecules; it will exaggerate the distances between bands. The disadvantage of higher concentrations is the long run times (sometimes days). Instead these gels should be run with a pulsed field electrophoresis (PFE), or field inversion electrophoresis.



Agarose ◦ Agarose is purified from agar, a gelatinous substance isolated from seaweed or red algae. Different purities of agarose are commercially available as are agaroses with different melting properties. High purity low melt agarose is often used if the DNA is to be extracted from the gel.



Buffer ◦ The most common being: tris acetate EDTA (TAE), Tris/Borate/EDTA (TBE) and Sodium borate (SB). TAE has the lowest buffering capacity but provides the best resolution for larger DNA. This means a lower voltage and more time, but a better product. SB is relatively new and is ineffective in resolving fragments larger than 5 kbp; However, with its low conductivity, a much higher voltage could be used (up to 35 V/cm), which means a shorter analysis time for routine electrophoresis.

An Agarose 'slab' gel prior to UV illumination, Only the marker dyes can be seen

The gel with UV illumination, the ethidium bromide stained DNA glows pink

Digital photo of the gel. Lane 1. Commercial DNA Markers (1kbplus)

Making an Agarose Gel

Agarose

D-galactose

3,6-anhydro L-galactose

•Sweetened agarose gels have been eaten in the Far East since the 17th century.

*Lina Hesse, technician and illustrator for  a colleague of Koch was the first to  suggest agar for use in culturing bacteria

•Agarose was first used in biology when Robert Koch* used it as a culture medium for Tuberculosis bacteria in 1882

Agarose is a linear polymer extracted from seaweed.

An agarose gel is prepared by combining agarose powder and a buffer solution.

Buffer➘

Flask for boiling➙

Agarose➚

Electrophoresis Equipment Power supply Cover

Gel tank

Electrical leads 

Casting tray Gel combs

Bio-Rad’s Electrophoresis Equipment

Electrophore sis Cells • Power Supplies • Precast Agarose Gels

PowerPac™ Junior

PowerPac™ HC

PowerPac™ Basic

PowerPac™ Universal

Mini-Sub® Cell GT

PowerPac™ 3000

Wide Mini-Sub Cell GT

Gel casting tray & combs

Preparing the Casting Tray

Seal the edges of the casting tray and put in the combs. Place the casting tray on a level surface. None of the gel combs should be touching the surface of the casting tray.

Agarose

Buffer Solution

Combine the agarose powder and buffer solution. Use a flask that is several times larger than the volume of buffer.

Melting the Agarose

Agarose is insoluble at room temperature (left). The agarose solution is boiled until clear (right). Gently swirl the solution periodically when heating to allow all the grains of agarose to dissolve. ***Be careful when boiling - the agarose solution may become superheated and may boil violently if it has been heated too long in a microwave oven.

Pouring the gel

Allow the agarose solution to cool slightly (~60ºC) and then carefully pour the melted agarose solution into the casting tray. Avoid air bubbles.

Each of the gel combs should be submerged in the melted agarose solution.

When cooled, the agarose polymerizes, forming a flexible gel. It should appear lighter in color when completely cooled (30-45 minutes). Carefully remove the combs and tape.

Place the gel in the electrophoresis chamber.

DNA buffer 

 wells

Cathode (negative)





 Anode (positive)

Add enough electrophoresis buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer.

Sample Preparation Mix the samples of DNA with the 6X sample loading buffer. This allows the samples to be seen when loading onto the gel, and increases the density of the samples, causing them to sink into the gel wells.

6X Loading Buffer:  • Bromophenol Blue (for color) • Glycerol (for weight)

Loading the Gel

Carefully place the pipette tip over a well and gently expel the sample. The sample should sink into the well. Be careful not to puncture the gel with the pipette tip.

Running the Gel

Place the cover on the electrophoresis chamber, connecting the electrical leads. Connect the electrical leads to the power supply. Be sure the leads are attached correctly - DNA migrates toward the anode (red). When the power is turned on, bubbles should form on the electrodes in the electrophoresis chamber.

Cathode (-)  wells  Bromophenol Blue

DNA (-) 

Gel Anode (+) After the current is applied, make sure the Gel is running in the correct direction. Bromophenol blue will run in the same direction as the DNA.

DNA Ladder Standard  12,000 bp  5,000

DNA migration Note: bromophenol blue migrates at approximately the same rate as a 300 bp DNA molecule bromophenol blue

 2,000  1,650  1,000  850  650  500  400  300  200  100

+ Inclusion of a DNA ladder (DNAs of know sizes) on the gel makes it easy to determine the sizes of unknown DNAs.

Staining the Gel • Ethidium bromide binds to DNA and fluoresces under UV light, allowing the visualization of DNA on a Gel. • Ethidium bromide can be added to the gel and/or running buffer before the gel is run or the gel can be stained after it has run.

***CAUTION! Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be worn at all times.

Safer alternatives to Ethidium Bromide • Methylene Blue • BioRAD - Bio-Safe DNA Stain • Ward’s - QUIKView DNA Stain • Carolina BLU Stain

…others advantages Inexpensive Less toxic No UV light required No hazardous waste disposal

disadvantages Less sensitive More DNA needed on gel Longer staining/destaining time

Staining the Gel

• Place the gel in the staining tray containing warm diluted stain. • Allow the gel to stain for 25-30 minutes. • To remove excess stain, allow the gel to destain in water. • Replace water several times for efficient destain.

Ethidium Bromide requires an ultraviolet light source to visualize

Visualizing the DNA (ethidium bromide) DNA ladder  1

2

3

4

5

6

7

DNA ladder 8 

wells

 5,000 bp  2,000  1,650  1,000  850  650  500  400  300  200  100

+

-

-

+

-

+

+

-

Visualizing the DNA (QuikVIEW stain)

wells

DNA ladder 

+ - - - - + + - - + - +

 2,000 bp  1,500  1,000  750  500  250



Uncut plasmid DNA has several distinct conformations which can be identified when the uncut plasmid is electrophoresed in an agarose gel.

◦ Supercoiled DNA (Form I), is the fastest conformation of the uncut plasmid. Because of its compact shape, sc DNA is the fastest moving comformation in the gel. ◦ Nicked Circle DNA (Form II) is also called relaxed circle. Nicked circle (nc) is the slowest conformation of uncut DNA. ◦ Linear DNA (Form III) is produced when a restriction enzyme cuts a plasmid at only one site. On a gel the linear DNA will run between the sc and nc conformations, often closer to the sc band

Figure 7­26 Copyright (c) by W. H. Freeman and Company



DNA can be recovered from agarose gel ◦ Electroelution : a slice of agarose containing DNA bend is placed in a dialysis bag inside a electrophoresis app. ◦ DNA bend is sliced agarose be melted in Na iodide buffer bound to amembran/ resin DNA is eluted ◦ Using low-melting agarose & +agarase ◦ Centrifugation-filtration ◦ Using QIAgen Gel Extraction kit

DNA FINGERPRINTING PROCEDURE

Molecular Weight Determination Fingerprinting Standard Curve: Semi-log Size (bp)

100,000

Distance (mm)

23,000 11.0 13.0

6,500

15.0

4,400

18.0

10,000

Size, base pairs

9,400

B

1,000

2,300

23.0

2,000

24.0 100 0

5

10

15

Distance, mm

20

A

25

30