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VIRAL DNA PURIFICATION “Trino’s Lab” Page 209-231

VIRAL DNA PURIFICATION “Trino’s Lab” Page 209-231. FAMILY: Geminiviridae Genus: Begomovirus Species: Tomato golden mosaic virus (TGMV ). Properties of DNA. Large polymers made up of nitrogenous bases and 5 carbon sugars linked by phosphodiester bonds.

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VIRAL DNA PURIFICATION “Trino’s Lab” Page 209-231

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  1. VIRAL DNA PURIFICATION “Trino’s Lab”Page 209-231 FAMILY: Geminiviridae Genus: Begomovirus Species: Tomato golden mosaic virus (TGMV)

  2. Properties of DNA • Large polymers made up of nitrogenous bases and 5 carbon sugars linked by phosphodiester bonds. • Nucleic acids because of phosphate groups with pKa of 1-2. Produces a strong acid with net – charge at neutral pH

  3. Amax Protein Average Amax is at A260

  4. Properties of DNA • BORING • But……………………..

  5. Relatively easy to Isolate • Obtained in very high molecular weight form. • DNA Molecule is designed to retain integrity • mRNA by contrast intended to be degraded • Protein subject to attack by enzymes and may be pH ionic strength and temperature sensitive • Proteins have personality • Different protocols are designed for different sources of DNA not differences in the molecule.

  6. GEMINIVIRAL DNA PURIFICATION • GEMINIVIRUSES: • Plant DNA viruses • ssDNA (most plant viruses are RNA) • Replication in nuclei • Whitefly transmitted • Problem worldwide

  7. GEMINIVIRAL DNA PURIFICATION • Geminiviruses are named by its shape under electron microscope

  8. GEMINIVIRAL DNA PURIFICATION • Four genus: • Mastrevirus • Curtovirus • Topocuvirus • Begomovirus: • Begomovirus are whitefly transmitted

  9. GEMINIVIRAL DNA PURIFICATION • Begomovirus infect dicot plant species, such as tomato, potato, tobacco, pepper, cassava, cotton and many others.

  10. Leaf Morphology Uninfected Infected

  11. GEMINIVIRUS DNA PURIFICATION • Begomovirus contain a circular ssDNA genome that can be mono or bipartite, of about 2.5 kb each. • TGMV (Tomato Golden Mosaic Virus) is a bipartite geminivirus • They code for only 6 genes. • Replicated by plant machinery in nuclei

  12. Big differences among animal and plant tissue can make difficult to extract nucleic acids. Cell wall Polysacharides Nucleases DNA POPULATIONS: Chromosomal DNA Mitochondrial DNA Chloroplast DNA Viral DNA GEMINIVIRUS DNA PURIFICATION

  13. Schedule • Day one:   First two pages of protocol( We will do the dry blot which will sit overnight)Day two:  U V Fixation Prepare the probe that will be used in day three. You will do   Hybridization ( The hybridization will run overnight)Day three: Complete Hybridization, detection and photo record. • Read introduction and theory Page 211-221 • Read protocol summary Page 222-223

  14. Day one: Extraction of DNA from infected leaf (Page 224) • One leaf infected; one uninfected • Grind by rotation while pressing down in 250 microliters of extraction buffer • After homogenized add buffer to total volume of 750 microliters • Mix well and wait 30 min

  15. Protocol • Very typical this basic approach can be used to purify smaller DNAs from virtually any source (Hirt extraction)and large RNAs from bacteria • Like the LDH purification begin by grinding tissue. • Conduct a chloroform/phenol extraction • Save aqueous phase • PPT the DNA with alcohol

  16. GEMINIVIRAL DNA PURIFICATION (Bench protocol Page 412) • Homogenize sample (with pestle) in buffer (step 1-4) • Incubate (10 min at 65C Step 5) • Centrifuge 2min (6)Save supernatent • Mix supernatant with phenol:chloroform (7) • Note: Protocol says add equal amount. A little more is better • Centrifuge 2 min (8) • Separate aqueous phase (9) • Isopropanol and salt precipitation (10) • Ethanol wash (step 12) • Dry and resuspend in dd water (Step 14)

  17. Gel separation of virus DNA • GEL PREPARATION AND RUNNING: • Agarose melting and mix with Ethidium Bromide (MUTAGENIC, WARNING !!!) • Pouring, wait until is solid. • Mix your DNA from purification with loading buffer and add to gel. • Set 120 Volts constant, run for 30 minutes • Take photographic register of the gel

  18. Denaturing and preparation of DNA for transfer to membrane • Denature DNA in gel (Step 1-5) • Neutralize (step 6-7) • Set up transfer to nylon membrane

  19. Transfer of DNA from gel to nylon membrane • SOUTHERN TRANSFER: • Cut the Nylon membrane to the same size of the gel (nylon is positively charged) • Cut filter paper and absorbent paper enough to make a 10cm pile (about 2-3 inches).

  20. Transfer of DNA from Gel to Membrane

  21. Allow Transfer to Occur Overnight End of Day One

  22. Day Two Membrane fixation, Probe preparation, Hybridization

  23. Gemini Virus DNA Gel M I I U U M M=marker, I= infected, U= uninfected, Arrow indicates virus RNA

  24. Preparation of probe and hybridization • UV Fixation (page 413 step 10) • Once the gel has been transferred (about 8-16 hours), take the Nylon membrane and expose it to UV light, using a crosslinking cabnet. • Set the crosslinker to optimal. • Instructor will do this step • TA will prepare probe • Week two: Detection, Probe with a SS DNA complementary to Gemini virus DNA (page 413 step 1-3)

  25. Probe preparation:Protocol • Produced an 800 BP long probe labeled by incorporation of NTPs labeled with digoxigenin.

  26. You will use probe to detect virus DNA in your blot (page 196 steps 1-9) This is a Southern hybridization DNA to DNA. Probe binds by base pairing with Denatured DNA on the nylon filter An antibody can detect the DU labled probe

  27. Hybridization • Requires extended time at moderate temperature • Selects for accurate hybrids • Leave overnight Page 414 step 5 • Ta will complete steps 6,7,8&9 • End of Day two

  28. Day Three Identification of Virus DNA in mixture of host cell DNA by antibody binding to DU labled Gemini specific probe

  29. Day three • Detection of virus DNA • Use DU specific antibody conjugate • Page 196 steps 1-8 • Record by digital photography step 9 &10

  30. Immuno-detection of virus specific probe

  31. From Last Year M=Not Infected I = Infected M I M I DS DNA SS DNA

  32. Week 3 • TA will complete steps 1&2 page 414 • Add 5ml of blocking buffer containing 1 microliter of antibody • Incubate 30 min • Pour into sink • Wash twice with 1X washing solution • Add 3 ml detection buffer • Rock for 4 minutes • Add 5ml of substrate • Wait 30min. Check for detection • Rx is complete in 5 hours

  33. This Lab • Three weeks • Report 40 points • Pre Lab 9 points

  34. Next Week • QPCR • Lab D3 Page 253 • Read introduction and Theory • Page 258-264 • Protocol page 266

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