DNA Technology: GEL ELECTROHPHORESIS

1. DNA Technology: GEL ELECTROHPHORESIS Ms. Day Honors Genetics

2. 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

3. Gel electrophoresis • Separates DNA restriction fragments of different lengths • Uses electrical currentto 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.

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

5. DdeI DdeI DdeI DdeI Normal  -globin allele 201 bp Large fragment 175 bp Sickle-cell mutant -globin allele Large fragment 376 bp DdeI DdeI DdeI (a) DdeIrestriction sites in normal and sickle-cell alleles of -globin gene. Sickle-cellallele Normalallele Largefragment 376 bp 201 bp175 bp (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles. Restriction Fragment Analysis • Is useful for comparing two different DNA molecules, such as two alleles for a gene • http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

6. Agarose Gel Used in Electrophoresis Widely used in gel electrophoresis technique for the analysis of DNA (or RNA or proteins) Routinely used (crime scenes, maternity/paternity cases, etc) Separates molecules based on their rate of movement through a gel under the influence of an electrical current Agarose gel is NOT agar but feels similar

7. Agarose • Agarose is extracted from seaweed

8. To separate a mixture of DNA fragments by size using an electrical charge The gel is a protein matrix (like a sponge with holes; DNA travels through “holes”) Purpose of Agarose Gel Electrophoresis • Agarose is porous, allowing for the movement of DNA Scanning Electron Micrograph of Agarose Gel (1×1 µm) 

9. How does gel electrophoresis separate DNA fragments? • Gel acts as a strainer to filter DNA by size • DNA fragments are naturally negatively charged due to the phosphate backbone (PO4-3) • DNA fragments of differing sizes will move though the gel at differing rates • larger fragments (more bases) = do not travel as far from wells • smaller fragments (less bases) = travel farther from wells

10. Movement depends on Charge • DNA is negatively charged (because of phosphate backbone) • DNA will be attracted to positively charged poles and repelled from negatively charged ones

11. DNA - + Power Movement Depends on Size • Small DNA move faster than larger pieces DNA • Gel electrophoresis separates DNA according to size • Power source supplies the electrical current small large Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.

12. Restriction Enzymes and Plasmid Mapping

13. Restriction Enzyme Digest  different length pieces are made

14. Gel electrophoresis markers (called standards or ladders) are used for size identification of each DNA fragment Each well/column is a “DNA fingerprint”

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

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

17. #1: Make Gel • combine agarose powder and buffer (ions + H2o) solution into a flask. Buffer Flask for boiling  Agarose

18. A. Agarose Buffer Solution Combine the agarose powder FIRST then and buffer solution.

19. Melting the Agarose B. Agarose is insoluble at room temperature (left). The agarose solution is boiled until clear (right). • Gently swirl the solution to dissolve. • Boil the solution • ***Be careful when boiling: agarose solution may become superheated and may boil OVER causing a STICKY MESS 

20. Gel casting tray & combs C. • Tray makes gel • Comb teeth makes wells

21. Preparing the Casting Tray C. COMBS CREATE WELLS!!!

22. Pouring the gel D. Allow the agarose solution to cool slightly (~60ºC) and then carefully pour the melted agarose solution into the casting tray. Avoid air bubble, why?

23. D. Make sure that the gel combs are submerged in the melted agarose solution but not touching the bottom.

24. E. When cooled, agarose forms a flexible gel. It appears cloudy in color when completely cooled (~20 minutes). Carefully remove comb (be very, very careful…don’t remove at an angle!).

25. Place the gel in the electrophoresis chamber.

26. DNA buffer     wells Anode (positive end) RED WIRE! Cathode (negative end) BLACK WIRE! Add enough buffer to cover the gel. Make sure each well is filled with buffer. Fxn: Buffer allows electrical current to FLOW through chamber!

27. Loading and Running Gel • After gel is made and place in chamber, DNA will be loaded into well THROUGH buffer. • 1st : DNA are cut into fragments using endonucleases • 2nd DNA samples are mixed w/ loading dye • FUNCTION: • weigh down DNA into wells • acts as a mobile dye so you can visualize migration • this is why DNA “falls” into wells and you can SEE it move through gel!!!

28. Sample Preparation • Samples of DNA need to be mixed with tracking dye. • Allows DNA samples to be seen in the gel • Increases the density of samples, causing them to sink into the gel wells. Loading Dye:  FUNCTIONS:  dye to track movement of DNA in gel (color)  Glycerol (for weight)

29. Loading the Gel Carefully place the micropipette tip over a well and gently expel the sample. The sample should sink into the well NOT float in the buffer. Be careful not to puncture the gel with the pipette tip.

30. Running the Gel • Place cover on electrophoresis chamber • Connect the electrical leads/wires • Be sure the leads are attached correctly - DNA migrates toward the anode (red). • Look for bubbles should form on electrodes when ON

31. Cathode (-) End  wells DNA (-)  Migration Tracking/loading dye Gel Anode (+) End After the current is applied, make sure the Gel is running in the correct direction. Loading dyewill run in the same direction as the DNA.

32. Staining the Gel • Ethidium bromide binds to DNA and fluoresces under UV light, allowing the visualization of DNA on a Gel. YOU ARE USING A QUICK DNA STAIN!!! •Function= allows you to SEE the bands after the gel is made ***CAUTION! Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be worn at all times.

33. 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.

34. 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.

35. Methylene blue requires an ultraviolet light source to visualize

36. Review- Know the functions of in Gel Electrophoresis • Restriction enzyme(s)= makes DNA fragments • Agarose gel= separates DNA fragments based on size • Comb= Makes wells to load DNA into • Casting tray= Make agarose gel • Loading dye= Weighs down DNA & tracks DNA movement through gel • Power source= supplies electrical current • Buffer= maintains current • Stain= makes DNA visible in gel

37. Visualizing the DNA (Actual Image) DNA ladder  wells  2,000 bp DNA  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

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

39. Semilog paper

40. GRAPH THE LADDER/STANDARD…then make a best fit line or curve! Fragment Length (bp) Distance migrated (mm)

41. x bp Fragment Length (bp) Distance migrated (mm)