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MB 206 : Module 2 - B

MB 206 : Module 2 - B. Extraction of Nucleic Acids (Genomic DNA, mRNA and Plasmid DNA ). Isolation of Nucleic Acids. Types of Methods : differential solubility ‘adsorption’ methods density gradient centrifugation. Goals : removal of proteins DNA vs RNA

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MB 206 : Module 2 - B

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  1. MB 206 : Module 2 - B Extraction of Nucleic Acids (Genomic DNA, mRNA and Plasmid DNA)

  2. Isolation of Nucleic Acids • Types of Methods: • differential solubility • ‘adsorption’ methods • density gradient centrifugation • Goals: • removal of proteins • DNA vs RNA • isolate specific type of nucleic acid • Types : • genomic (chromosomal) • organellar (satellite) • plasmid (extra-chromosomal) • phage/viral (ds or ss) • complementary (mRNA)

  3. Extraction of total genomic DNA • Plant, animal or bacteria cells, tissues (spleen, liver), whole organisms. • Pretreatment depends on the nature of cell wall present. • No pretreatment for mammalian cells & E.coli - simple nuclei lysis. • Gram-positive bacteria cells have thick cell wall need pretreatment - digestion of cell wall with lysozymes. • Genomic DNA purification kit are available to isolate genomic DNA

  4. Nucleic Acid Extraction Requirements • Extraction/Precipitation method • Adsorption Chromatography method

  5. Getting Prepared: Creating a Nuclease-Free Environment Important There are several things you can do to minimize the risk of exposing your samples to external DNases and RNases. • Autoclave solutions. This is usually sufficient for getting rid of DNases, and most RNases as well. •Treat solutions with 0.1% DEPC. DEPC inactivates nucleases by covalently modifying the His residues in proteins. Generally considered unnecessary for DNA extraction. Not compatible with solutions containing Tris or HEPES. • Have a dedicated set of pipettors or use aerosol barrier tips. • Wear gloves (especially for RNA). You should be doing this anyway for safety reasons, but skin cells also produce RNases, a potent RNA-degrading enzyme. • Bake glass, metal, or ceramic equipment at high temp.

  6. Cells Extract Bacterial Cells Or tissue culture cells Or blood Or flies……….. HOW? Organic extraction Pure DNA

  7.  aqueous phase (nucleic acids)  phenol phase (proteins) High MW Genomic DNA Isolation • Detail of step 3 • Phenol Extraction • mix sample with equal volume of sat. phenol soln • retain aqueous phase • optional chloroform/isoamyl alcohol extraction(s) • Typical Procedure • Harvest cells • 2 Cell Lysis • 0.5% SDS + proteinase K (55o several hours) • 3 Phenol Extraction • gentle rocking several hours • 4 Ethanol Precipitation • 5 RNAse followed by proteinase K • 6 Repeat Phenol Extrac-tion and EtOH ppt

  8. Extraction/Precipitation Method Step 3:Organic extraction Mix thoroughly with an equal volume of organic solvent Aqueous Centrifuge Collect aqueous phase e.g. phenol, chloroform, or phenol:chloroform Interphase Organic Perform additional extractions for increased purity Crude lysate containing nucleic acids and other cell constituents The aqueous phase contains water-soluble molecules, including nucleic acids. Proteins and lipids become trapped in the organic phase, and are thus separated away. Insoluble debris become trapped in the interphase between the two layers

  9. High MW Genomic DNA Isolation • Detail of step 4 • EtOH Precipitation • 2-2.5 volumes EtOH, -20o • high salt, pH 5-5.5 • centrifuge or ‘spool’ out • Typical Procedure • Harvest cells • 2 Cell Lysis • 0.5% SDS + proteinase K (55o several hours) • 3 Phenol Extraction • gentle rocking several hours • 4 Ethanol Precipitation • 5 RNAse followed by proteinase K • 6 Repeat Phenol Extrac-tion and EtOH ppt

  10. Extraction/Precipitation Method Step 4:Nucleic Acid Precipitation Before After After Supernatant 70% EtOH Centrifuge Wash Centrifuge Pellet Dissolve pellet (H2O, TE, etc.) • Pellet down nucleic acids. • Pellet down nucleic acids. • Wash pellet with 70% ethanol to remove residual salts and other contaminants. • Pellet down nucleic acids. • Wash pellet with 70% ethanol to remove residual salts and other contaminants. • Discard ethanol and allow pellet to dry. Add alcohol and salt to precipitate nucleic acids from the aqueous fraction

  11. Detail of step 5 Using Nucleases to Remove Unwanted DNA or RNA Add DNase + DNase (protein) Add RNase + RNase (protein) Depending on when nuclease treatment is performed, it may be necessary to repeat purification steps for protein removal (e.g. phenol/chloroform extraction).

  12. Preparing genomic DNA from whole organisms (eg. Drosophila) / tissues 1. Homogenization: whole organisms / tissue - snap frozen in liquid Nitrogen using a mortar and pestle. 2. Cell and Nuclei Lysis: resuspend homogenized material in nuclei lysis Buffer (Tris HCL, EDTA, 1% SDS). 3. Digestion with Proteinase K: to degrade proteinaceous components (100 mg/ml for 1-2h @ 37oC with occassional swirling). 4. Phenol-Chloroform extraction: to remove degraded proteins (forms a while interphase between top aqueous layer (contain DNA) and the bottom organic layer (phenol/chloroform). 5. Precipitate the DNA: from the aqueous phase by adding half vol 7.5M NH4OAc and equal vol of isopropanol; mix gently, leave o/n @ -20oC 6. Pellet DNA: by centrifuging and rinse DNA pellet in 70% EtOH and air dry briefly 7. Resuspend genomic DNA in TE buffer. 8. RNase A treatment: @ 100mg/ml; 37oC, 30 min 9. Repeat phenol-chloroform extraction to remove residue RNase and NaOAc/EtOH ppt. 10. Store DNAin Tris-EDTA buffer (pH 8.0), at 4oC (short term) or 20oC (long term)

  13. Preparing genomic DNA from cells or tissues using commercially available kits (eg. Qiagen / Promega) • Harvest cells / tissues (homogenize) • Add Nuclei Lysis Solutionand pipette to lyse the cells - no visible cell clumps remain • 3. Treat nuclear lysate with RNase Solution, mix the sample by inverting the tube 2.5 times (15 min at 37°C) • 4. Add Protein Precipitation Solutionand vortex vigorously at high speed for 20 sec. • 6. Centrifuge to pellet the precipitated protein (white pellet) • 7. Carefully separate the supernatant containing the DNA • 8. Isopropanol precipitation of DNA can be replaced by placing the supernatant onto a spin column, DNA captured on the positively charged silica column, washed and eluted in TE buffer/ H2O.

  14. Gregor Mendel (1822-1884) Thomas H. Morgan 1933, Nobel prize for his study of fruit flies Paper in 1860 1920s-1940: Embryologists observe that there arehereditary determinants in the cytoplasm. 1950s: reported thatcytoplasmic hereditary units in yeast mitochondria, and in the chloroplast of Chlamydomonas. Plasmid Early History Time-Line: 1903: Walter S. Sutton and Theodor Boveri independently hypothesize that the units of Mendelian characters are physically located on chromosomes. 1910: Thomas Hunt Morgan (1866-1945) describes association of genes with a specific chromosome in the nucleusof Drosophila.

  15. Plasmid Early History continued 1946 -1951: Joshua Lederberg et al., report strong evidence for a sexual phase in E. coli K-12. Meanwhile, lysogenic phages were also studied. 1950-1952: William Hayes suggests that mating in E. coli is an asymmetric (unidirectional) process. 1952: J. Lederberg reviews the literature on cell heredity and suggests the term "Plasmid" for all extrachromosomal hereditary determinants. 1952-1953: W. Hayes, and J. Lederberg, Cavalli, and E. Lederberg report that the ability to mate is controlled by a factor (F)that seems to be not associated with the chromosome. ( in the summer of 1952: James D. Watson described the event (The Double Helix)). Schematic drawing of bacterial conjugation. 1, Chromosomal DNA. 2, F-factor (Plasmids). 3, Pilus.

  16. Plasmid Early History continued 1954: Pierre Fredéricq and colleagues show that colicine (plasmids) (large toxin proteins (50-70kD)) behave as genetic factors independent of the chromosome. 1958: François Jacob and Elie Wollman propose the term "Episome" to describe genetic elements such as F factor, colicine, and phage lambda, which can exist both in association with the chromosome and independent of it. 1961: DNA (radioactive) labeling show that mating in bacteria is accompanied by transfer of DNA from the donor to the recipient. 1962: In a review on episomes, Allan Campbell proposes the reciprocal recombination of circular episome DNA molecules with the chromosomal DNA. 1962: Circular DNA is found to actually exist in the genome of the small phage phi-X174.

  17. Work with Plasmid DNAs Isolation and Purification Banding of plasmids and chromosomal DNAs in CsCl-EtBr and in iodixanol-DAPI gradients. After 10 hrs centrifugation at 100,000 rpm (450,000 xg), two distinct bands, corresponding to linear nuclear DNA above and circular mitochondrial DNA below, are visible under ultraviolet light. CsCl Gradient centrifugation or CsCl dye-bouyant density method

  18. Separation of Nucleic Acids by CeCl Gradient Centrifugation

  19. Plasmid DNA

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