DNA Technology: GEL ELECTROHPHORESIS Ms. Day AP Biology

DNA Gel Electrophoresis DNA fingerprint **Each band that you see is a collection of millions of DNA molecules, all of the same length!! Restriction Fragment Analysis detects DNA differences that affect restriction sites

What is Gel Electrophoresis? Process that separates DNA restriction fragments of different lengths DNA cut with restriction enzymes Uses electrical current to separate DNA based on size DNA has a negative charge. DNA moves towards the POSITIVE electrode. Why? DNA molecules of SMALLER sizes move the furthest through the gel.

Movement depends on Charge DNA is negatively charged (because of phosphate backbone)

Purpose of Agarose Gel Electrophoresis: Gel electrophoresis separates a mixture of DNA fragments according to size Uses an agarose gel to do this Made of sugar from seaweed Agarose gel acts as a strainer to filter DNA fragments based on size a gel contains a protein matrix It is porous Has “holes” like a sponge Now it would be great to make a picture of many lines of various sizes mixed up on one size and organize according to size in the other Scanning Electron Micrograph of Agarose Gel (1×1 µm) 

Agarose: Agarose is a linear polymer extracted from seaweed. D-galactose 3,6-anhydro L-galactose Sweetened agarose gels have been eaten in the Far East since the 17th century. Agarose was first used in biology when Robert Koch* used it as a culture medium for Tuberculosis bacteria in 1882 Can be used to separate DNA fragments > 300 bp Agarose is a linear polymer extracted from seaweed.

http://www. sumanasinc http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html

How does gel electrophoresis separate DNA fragments? DNA fragments of differing sizes will move though the gel at differing rates (speeds) larger fragments = more bases  do not travel as far as smaller fragments containing less bases Larger fragments get “stuck” in gel sooner than smaller pieces

Movement Depends on Size: Small DNA move faster than large DNA …gel electrophoresis separates DNA according to size DNA + - Power small large Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.

Gel Electrophoresis Equipment Power supply Cover Gel tank Electrical leads  Casting tray Gel combs

Electrophoresis Apparatus Making an Agarose Gel And Setting up your Gel Electrophoresis Apparatus

ions in aqueous solution. Combine: agarose powder a buffer ions in aqueous solution. Buffer Flask for boiling Agarose

Use a flask that is several times larger than the volume of buffer. Agarose Buffer Solution Use a flask that is several times larger than the volume of buffer.

Agarose is insoluble (cloudy) at room temp Melting the Agarose Agarose is insoluble (cloudy) at room temp Agarose solution is boiled until clear (right). Gently swirl the solution ***Be careful when boiling agarose solution may become superheated and boil violently over  A SUGARY MESS 

Gel casting tray & combs

Preparing the Casting Tray Seal edges of casting tray with black plastic sides Put in 1 comb with teeth  will create wells for DNA samples Place the casting tray on a level surface Pour molted gel into casting tray BE CAREFUL  HOT!!!

Avoid air bubbles when pouring (“casting”) gels, why? Pouring the gel Avoid air bubbles when pouring (“casting”) gels, why?

Make sure gel comb teeth are submerged in melted agarose solution…why?

When cooled, the agarose polymerizes, forming a flexible gel When cooled, the agarose polymerizes, forming a flexible gel. It should appear lighter in color when completely cooled (30-45 minutes). Carefully remove the comb (be very, very careful!).

Making Wells… After agarose solidifies, comb is removed leaving wells where the DNA will be loaded. Remove comb by pulling up at 90° angle Comb teeth create wells, where DNA is placed

Place the gel in the electrophoresis chamber.

Add buffer to cover the gel to a depth of at least 1 mm. DNA buffer   wells    Anode (positive end) RED WIRE! Cathode (negative end) BLACK WIRE! Add buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer.

DNA Sample Preparation 1. DNA samples are digested using restriction enzymes  Fragments are made! 2. Mix the samples of DNA with loading solution (w/ tracking dye). This allows: DNA samples to be seen when loading and running gel Increases density of DNA samples, causing them to sink into the gel wells. Mixture contains…  Bromophenol Blue (for color)  Glycerol (for weight)

Loading the Gel Carefully place the pipette tip over a well Gently expel DNA sample. The sample should sink into the well. Be careful not to puncture the gel with the pipette tip.

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

Cathode (-) End DNA (-)  Migration  wells  Bromophenol Blue Gel Anode (+) End After current is applied, make sure the gel is running in the correct direction. Tracking dye will run in the same direction as the DNA just ahead of it

**STAIN is different than LOADING DYE Staining the Gel • Stain binds to DNA and fluoresces under UV light, allowing visualization of DNA bands on gel. BUT…..YOU ARE USING A QUICK DNA STAIN!!! **STAIN is different than LOADING DYE ***CAUTION! Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be worn at all times.

• Place the gel in the staining tray Staining the Gel • Place the gel in the staining tray Slide it in from casting tray AFTER gel is run

Staining the Gel • Place the gel in the staining tray containing warm diluted stain. • Allow the gel to stain for 15-20 minutes. • To remove excess stain, allow the gel to destain in water. • Replace water several times for efficient destain.

Methylene blue requires an ultraviolet light source to visualize

Visualizing the DNA  100  200  300  1,650  1,000  500  850  650  400 5,000 bp  2,000 DNA ladder  DNA: 1 2 3 4 5 6 7 8 wells + - - + - + + - Samples # 1, 4, 6 & 7 were positive for DNA samples taken from the crime and compared to suspect

Samples # 1, 6, 7, 10 & 12 were positive for our suspect and Visualizing the DNA (Actual Image) DNA ladder  wells DNA  2,000 bp  1,500  1,000  750  500  250 + - - - - + + - - + - + Samples # 1, 6, 7, 10 & 12 were positive for our suspect and crime scene samples March 12, 2006

Movement of DNA fragments in agarose gels There is a linear relationship between” the migration rate of a DNA fragment and The size of the fragment (in basepairs). Larger molecules move more slowly through the gel because of more friction

Semilog paper

You have to graph the STANDARD marker DNA fingerprint on the semilog paper Fragment Length (bp) Distance migrated (mm)

Then you USE the graph to find the UNKNOWN fragment lengths! x bp this is the length of the unknown fragment Fragment Length (bp) Distance migrated (mm)