EXPERIMENT GENOMIC DNA ANALYSIS Population diversity Studies We have 5 species of planarians (3 purchased from Carolina Biologicals, 2 obtained from the Levin lab) andmight have additional species found in ponds in Massachusetts. How genetically related are those planaria to each other? And how related are planaria of the same species? Unless two organisms are clones of each other they are not genetically identical and will have some level of sequence diversity. DNA sequencing is the ultimate method of detecting single base differences. However this method is completely beyond the scope of this workshop. In lieu of DNA sequencing we will extract genomic DNA from individual planarians species, digest with specific restriction enzymes. and use gel electrophoresis to compare the pattern of digestions to determine how close the planaria are to each other genetically. This is the simplest way to rapidly determine if two worms are clones of each other or are different. a) Extracting genomic DNA Material: 1M Tris-HCl ph 8.0 0.5M EDTA, ph 8.0 20 mg/ml proteinase K Distilled sterile water 10% SDS Phenol/chloroform/isoamyalcohol (25:24:1) 3M Sodium Acetate 100% Ethanol (in freezer) 70% Ethanol TE RNAse A 20 mg/ml 1.5 ml microfuge tubes microcentrifuge Heat block at 65ºC Set of Pipettemans and tips Ice bucket Procedure: Keep everything on ice when not in heat block. Wear gloves! 1. Prepare lysis buffer. In a microfuge tube add: 395 µl of distilled sterile water
50 µl 1 M Tris-Hcl ph 8.0 25 µl 10% SDS 25 µl 0.5M EDTA, ph 8.0 5 µl of proteinase K Place on ice. 2. Transfer several worms (5 large) into an microfuge tube. Using a pipette, take out as much of the water as possible. Place in ice bucket. 3. Transfer the lysis buffer (500 µl) into the microfuge tube containing the worms. Do not vortex to avoid breaking the DNA. Mix by taping with finger or by inverting tube couple of times 4. Mash worms using a small blue plastic pestle. 5. Incubate at 65ºC for 2-3 hours. Invert tube couple times after 30 minutes incubation. The worm should disintegrate completely and the solution should become slightly brownish depending on the worm s color ******** LECTURE + LUNCH ********** 6. Add 500 µl (equal volume) of phenol/chloroform/isoamylalcohol. 7. Mix well by inverting back and forth. Do not vortex. The solution should become milky. 8. Microcentrifuge at top speed for 2 minutes 9. Using a P1000 pipetteman set at 500 µl transfer gently the top aqueous phase (should be kind of clear) to a new labeled tube. Avoid pipetting the interphase which contains denatured proteins. The volume that you transfer to the new tube should be about 450 µl due to loss of aqueous material at the interphase.. 10. Add 1/10 volume of 3 M sodium acetate. If the volume you transferred was for example 450 µl add 45 µl of 3 M sodium acetate. The final concentration of sodium acetate will be 0.3 M. 11. Mix by holding the tube between the index and thumb and inverting the tube several times. 12. Add 2 volumes of 100% ice cold ethanol (~ 900µl). Invert gently back and forth. If there is plenty of DNA you may see the DNA come out of solution as a white or black cotton ball (probably pigments) 13. Quickly close the tip of a glass Pasteur pipette by flaming, cool and use to spool up the DNA blob. 14. Immerse the spooled DNA into a microfuge tube containing 70% EtOH, swirl around or up and down to wash away the salt. 15. Let DNA air dry on Pasteur pipette on the bench for ~ 15 minutes by placing the end of the Pasteur pipette into a tube holder point up! The DNA may switch from white to transparent as it dries. 16. Immerse Pasteur pipette into a microfuge tube containing 200 µl sterile water; the DNA will dissolve in the water (you may have to scrape a little to get it all off the pipette) 17. Next add 5 µl of 0.5 mg/ml RNAse A. Incubate for 30 minutes at 37 ºC. 18. Add 300 µl of TE and repeat steps 6-15 as described above. 19. Resuspend in 50 µl of TE
20. Store DNA on ice or in freezer b) Digestion with restriction enzymes Choose 2 restriction enzymes that are known to cut well and are cheap (Hind III or EcoRI, and PAC I or Not I)). Hind III and EcoRI recognize a 6-base palindromes (six cutters) whereas PAC I and Not I recognize an 8 -base palindromes. The probability that a specific n bases sequence is found randomly is 1 in every 4 n bases. To set up a digest: 1. For each restriction enzyme label a microfuge tube with the restriction enzyme and the planaria (and date and initials) 2. Each DNA digest will be done in a total volume of 40 µl in a 1.5 ml microfuge tube Add to a tube on ice: 19 µl of distilled sterile water 15 µl of DNA sample 4 µl of 10X buffer (specific for each restriction enzyme) 2 µl of restriction enzyme 3. Incubate in a heat block at 37 ºC for one to 3 hours. 4. After the digest is done you can store the tubes in the freezer at 20ºC until ready to analyze. c) Analysis of digested genomic DNA by agarose gel electrophoresis Material Agarose TAE buffer Gel box, comb, and power supply Microcentrifuge 1. To prepare 1% agarose gels in 1X TAE. Weigh 1 g of agarose Add 100 ml of 1 XTAE buffer to a 250 ml erlenmeyer flask Add 1 g of agarose to the TAE Microwave 1.5 to 3 minutes to completely dissolve the agarose. Use thick protective gloves to remove the flask from the oven. Swirl around to mix. Avoid creating bubbles while mixing. If the agarose is fully dissolved the solution should be clear. Let stand on the bench while you are taping the tray. 2. Insert the gel tray side ways in the chamber to seal the side or use tape to seal the ends. Insert the comb. 3. When the agarose solution is around 55 C, add 10 µl of SYBR safe solution, mix gently and pour the solution into the tray until it reaches 3/4 of the teeth height. Make sure the tray is on a flat surface. Let solidify for at least 15 minutes. The solidified gel will be white and solid to the touch. Remove tape
from sides of tray. Remove comb. Set the tray correctly in the electrophoresis chamber with the teeth on the cathode side. Pour 1X TAE buffer in the chamber until all the wells are covered. 4. Retrieve DNA samples from freezer, thaw and put in ice bucket. 5. Add 5 µl of 10X loading dye to each sample. All samples will be loaded on the same gel. We will run duplicate gels. 6. Load 8 µl of Mw standards ( 1 Kb ladders) in the end lanes. Load the negative control (uncut DNA) on the second well from the left. Load 25 µl of each sample into the wells. 7. Close lid and connect leads to power supply. Apply 100 V. Run for 30 minutes to one hour. 8. Shut down power supply. Disconnect the leads. Take the tray out. HOLD the open sides of the tray to prevent the gel from slipping away. 9. Transfer the gel onto the UV box. Turn the UV light on and close the lid to view the DNA bands. 10. To take a picture, open the lid, Set the camera on top of the box and squeeze trigger. Pull out picture and let develop for 15 seconds. Use yellow filter. d) Analysis of results Using a ruler and semi log graph paper draw the Mw standard curve. For each planaria species how many bands are in the HindIII lane? How many bands in the PacI lane? What is the size of the largest band in each lane? Is the digest complete for each enzyme? Can you estimate the size of the genome for each species? What do the restriction patterns show in respect to the genetic similarity between the various planaria samples?