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Dna2014

  1. 1. By Dr Khaled Saleh Algariri
  2. 2. Every living thing has DNA. That means that you have something in common with a zebra, a tree, a mushroom and a beetle!!!! DNA stands for: D: Deoxyribose N: Nucleic A: Acid DNA is too small to see, but under a microscope it looks like a twisted up ladder!
  3. 3. Definition  DNA Extraction is the isolation and purification of DNA (deoxyribonucleic acid)
  4. 4. Examples  DNA extraction is used to isolate…  Mitochondrial DNA  Genomic DNA  DNA can be extracted from…  Cells or tissues  Environmental samples http://www.davidkfaux.org/shetlandislandsmtDNA.html http://faculty.uca.edu/~benw/biol1400/pictures/
  5. 5. Non-examples  DNA Extraction is not used to…  Isolate proteins or RNA  Give information about gene expression http://iwrwww1.fzk.de/biostruct/
  6. 6. Nucleic Acid Preparation Application?  DNA  Purity and amount of DNA required (and process used) depends on intended application. Example applications:  Tissue typing for organ transplant  Detection of pathogens  Human identity testing  Genetic research  Reverse transcription polymerase chain reaction and Ligase chain reaction(PCR, LCR)  RFLP (restriction fragment length polymorphism)  Hybridization methods (Southern analysis)
  7. 7. Nucleic Acid PreparationApplication?  RNA  Amplification methods , Reverse transcription polymerase chain reaction(RT-PCR)  Hybridization methods (Northern analysis)
  8. 8. DNA Purification Challenges 1. Separating DNA from other cellular components such as proteins, lipids, RNA, etc. 2. Avoiding fragmentation of the long DNA molecules by mechanical shearing or the action of endogenous nucleases. Effectively inactivating endogenous nucleases (DNase enzymes) and preventing them from digesting the genomic DNA is a key early step in the purification process. DNases can usually be inactivated by use of heat or chelating agents.
  9. 9. Nucleic Acid Purification There are many DNA purification methods. All must: 1. Effectively disrupt cells or tissues (usually using detergent) 2. Denature proteins and nucleoprotein complexes (a protease/denaturant) 3. Inactivate endogenous nucleases (chelating agents) 4. Purify nucleic acid target away from other nucleic acids and protein (could involve RNases, proteases, selective matrix and alcohol precipitations)
  10. 10. Disruption of Cells/Tissues Most purification methods disrupt cells using lysis buffer containing: Detergent to disrupt the lipid bilayer of the cell membrane Denaturants to release chromosomal DNA and denature proteins Additional enzymes are required for lysis of some cell types: Gram-positive bacteria require lysozyme to disrupt the bacterial cell wall. Yeasts require addition of lyticase to disrupt the cell wall. Plant cells may require cellulase pre-treatment.
  11. 11. Disruption of Cells: Membrane Disruption  Detergents are used to disrupt the lipid:lipid and lipid:protein interactions in the cell membrane, causing solubilization of the membrane.  Ionic detergents (such as sodium dodecyl sulfate; SDS) also denature proteins by binding to charged residues, leading to local changes in conformation.
  12. 12. Protein Denaturation Denaturation = Modification of conformation to unfold protein, disrupting secondary structure but not breaking the peptide bonds between amino acid residues. Denaturation results in:  Decreased protein solubility  Loss of biological activity  Improved digestion by proteases  Release of chromosomal DNA from nucleoprotein complexes (“unwinding” of DNA and release from associated histones)
  13. 13. Inactivation of Nucleases  Chelating agents, such as EDTA, sequester Mg2+ required for nuclease activity.  Proteinase K digests and destroys all proteins, including nucleases.  Some commercial purification systems provide a single solution for cell lysis, protein digestion/denaturation and nuclease inactivation.
  14. 14. Removal of RNA  Some procedures incorporate RNase digestion during cell lysate preparation.  In other procedures, RNase digestion is incorporated during wash steps.
  15. 15. Basic Protocol  Most DNA extraction protocols consist of two parts 1. A technique to lyse the cells gently and solubilize the DNA 2. Enzymatic or chemical methods to remove contaminating proteins, RNA, or macromolecules  In plants, the nucleus is protected within a nuclear membrane which is surrounded by a cell membrane and a cell wall. Four steps are used to remove and purify the DNA from the rest of the cell. 1. Lysis 2. Precipitation 3. Wash 4. Resuspension
  16. 16. A comparison of DNA extraction methods used in research labs as opposed to classroom labs Research Lysis: grind in Liquid N2 and use detergent Precipitation Part I: phenol/chloroform extraction to get rid of proteins Precipitation Part II: addition of salts to interrupt hydrogen bonding between water and phosphates on the DNA Precipitation Part III: addition of ethanol to pull DNA out of solution Wash and resuspend: DNA is washed in ethanol, dried, and resuspended in H20 or TE buffer. Classroom Lysis: grind in mortar/pestel and use detergent Precipitation Part I: NONE (chemical are too dangerous!) Precipitation Part II: addition of salts to interrupt hydrogen bonding between water and phosphates on the DNA Precipitation Part III: addition of ethanol to pull DNA out of solution Wash and resuspend: DNA is washed in ethanol, dried, and resuspended in H20 or TE buffer.
  17. 17. LYSIS: In DNA extraction from plants, this step commonly refers to the breaking of the cell wall and cellular membranes (most importantly, the plasma and nuclear membranes)  The cell wall (made of cellulose) is disrupted by mechanical force (for example, grinding the leaves)  Then the addition of a detergent in the which breaks down the cell membranes  Detergents are able to disrupt membranes due to the amphipathic (having both hydrophilic and hydrophobic regions) nature of both cellular membranes and detergent molecules. The detergent molecules are able to pull apart the membranes
  18. 18. DNA purification: phenol/chloroform extraction 1:1 phenol : chloroform or 25:24:1 phenol : chloroform : isoamyl alcohol Phenol: denatures proteins, precipitates form at interface between aqueous and organic layer Chloroform: increases density of organic layer Isoamyl alcohol: prevents foaming
  19. 19. PRECIPITATION (In a research lab): This a series of steps where DNA is separated from the rest of the cellular components  In a research lab, the first part of precipitation uses phenol/chloroform to remove the proteins from the DNA  Phenol denatures proteins and dissolves denatured proteins.  Chloroform is also a protein denaturant THIS STEP CANNOT BE PERFORMED IN CLASSROOM LABS!!  The second part of research lab DNA precipitation is the addition of salts  The salts interrupt the hydrogen bonds between the water and DNA molecules.  The DNA is then precipitated from the protein in a subsequent step with isopropanol or ethanol  In the presence of cations, ethanol induces a structural change in DNA molecules that causes them to aggregate and precipitate out of solution.  The DNA is pelleted by spinning with a centrifuge and the supernatant removed
  20. 20. PRECIPITATION (In a classroom lab): This a series of steps where DNA is separated from the rest of the cellular components  In a classroom lab, DNA precipitation involves the addition of salts  The salts interrupt the hydrogen bonds between the water and DNA molecules.  The DNA is then precipitated from the protein in a subsequent step with isopropanol or ethanol  In the presence of cations, ethanol induces a structural change in DNA molecules that causes them to aggregate and precipitate out of solution.  The DNA is pelleted by spinning with a centrifuge and the supernatant removed Note: because this protocol does not use phenol/chloroform, the DNA extracted in a classroom lab is not as “clean” as the DNA extracted in a research lab!
  21. 21. Washing: The precipitated DNA is laden with acetate salts. It is “washed” with a 70% ethanol solution to remove salts and other water soluble impurities but not resuspend the DNA. Resuspension: The clean DNA is now resuspended in a buffer to ensure stability and long term storage. The most commonly used buffer for resuspension is called 1xTE Washing and Resuspension:
  22. 22. cell growth cell harvest and lysis DNA purification DNA concentration
  23. 23. Bacterial genomic DNA prep: cell extract Lysis: • Detergents • Organic solvent • Proteases (lysozyme) • Heat “cell extract”
  24. 24. Genomic DNA prep: removing proteins and RNA Add the enzyme RNase to degrade RNA in the aqueous layer Need to mix gently! (to avoid shearing breakage of the genomic DNA) chloroform
  25. 25. 2 ways to concentrate the genomic DNA 70% final conc. “spooling” Ethanol precipitation
  26. 26. Genomic DNA prep in plants -- how get rid of carbohydrates? CTAB:cetyltrimethyla mmonium bromide, hexadecyltrimethylam monium bromide. Cationic detergent (low ionic conditions) N+ CH3 Br- CH3 CH3 C16H33
  27. 27. o Successful RNA isolation depends on: 1) Suppression of endogenous RNAases. 2) Avoid contamination with exogenous RNAases during extraction. A. Samples should be processed immediately or stored at - 70 degree until required. B. Inactivation of RNAases by strong denaturing agents like urea, guanidinium hydrochloride, guanidinium isothiocyanate. Suppression of endogenous RNAases
  28. 28. A. Specify glassware, solutions, equipments to be used for RNA extraction only. B. Treat water and laboratory utensils with diethylpyrocarbonate (DEPC) which is a strong RNAase inhibitor. DEPC is a suspected carcinogen. C. Autoclave glassware, solutions and equipments if possible. D. Use disposable gloves, disposable plastic materials that must be RNAase free. Avoid contamination with exogenous RNAases during extraction
  29. 29. 1) Guanidinium isothiocyanate extraction: Cell lysis. Protein denaturation by guanidinium isothiocyanate. Cell lysate is mixed with cesium chloride. The density of RNA in cesium chloride is much greater than of other cellular elements. During ultracentrifugation, RNA pellets at the bottom of the tube and becomes separated from other cellular components. Methods of RNA extraction
  30. 30. 2) RNA extraction by Trizol: Tizol is a monophasic solution of phenol & chloroform + guanidinium isothiocyanate. The presence of phenol & chloroform will separate cell lysate into two layers: o Upper aqueous layer containing RNA. o Organic layer containing proteins. RNA is then precipitated from the aqueous layer by isopropyl alcohol. 3) RNA extraction by spin column: These columns use RNA adsorbing silica or glass fiber. RNA is then eluted by elution buffer.
  31. 31. RNA extraction by Trizol
  32. 32. 4) RNA extraction by magnetic separation technology: Couple magnetic beads to silica. Magnetic silica beads binds RNA in the lysate. The conjugated magnetic beads are then collected by applying magnetic field. RNA is then eluted from the beads.
  33. 33. Separate WBCs from RBCs, if necessary Lyse WBCs or other nucleated cells in presence of protein denaturants, RNase inhibitors Denature/digest proteins Separate proteins, DNA, and contaminants from RNA Precipitate RNA if necessary Resuspend RNA in final buffer Basic Steps in Isolating RNA from Clinical Specimens
  34. 34. RNAses ►RNases are naturally occurring enzymes that degrade RNA ►Common laboratory contaminant (from bacterial and human sources) ►Also released from cellular compartments during isolation of RNA from biological samples ►Can be difficult to inactivate
  35. 35. RNAses ►RNAses are enzymes which are small proteins that can renature and become active. ►MUST be eliminated or inactivated BEFORE isolation.
  36. 36. Protecting Against RNAse ►Wear gloves at all times ►Use RNase-free tubes and pipet tips ►Use dedicated, RNase-free, chemicals ►Pre-treat materials with extended heat (180 C for several hours), wash with DEPC-treated water, NaOH or H2O2 ►Supplement reactions with RNase inhibitors
  37. 37. Total RNA ►80-90% of total RNA is ribosomal RNA. ►2.5-5% is messenger RNA ► 15-20% is transfer RNA
  38. 38. 2–25 °C 2–8 °C –20 °C –70 °C Recommended for long-term storage in ethanol <4 Months 1–3 Years <7 Years >7 Years Nucleic Acid Storage Requirements: Storage of DNA Specimens
  39. 39. BEST WISHES

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