1. Isolation of Nucleic Acids Goals: removal of proteins DNA vs RNA isolation of a specific type of DNA (or RNA) Types of Methods: differential solubility ‘adsorption’ methods density gradient centrifugation Types of DNA: genomic (chromosomal) organellar (satellite) plasmid (extra- chromosomal) phage/viral (ds or ss) complementary (mRNA) General Features: denaturing cell lysis (SDS, alkali, boiling, chaotropic)  enzyme treatments ­protease ­RNase (DNase-free) ­DNase (RNase-free) 1Dr.Saba Abdi

2. High MW Genomic DNA Isolation Typical Procedure 1Cell Lysis –0.5% SDS + proteinase K (55 o several hours) 2Phenol Extraction –gentle rocking several hours 3Ethanol Precipitation 4RNAse followed by proteinase K 5Repeat phenol extrac- tion and EtOH ppt Phenol Extraction mix sample with equal volume of sat. phenol soln retain aqueous phase optional chloroform/isoamyl alcohol extraction(s)  aqueous phase (nucleic acids)  phenol phase (proteins) 2Dr.Saba Abdi

3. High MW Genomic DNA Isolation Typical Procedure 1Cell Lysis –0.5% SDS + proteinase K (55 o several hours) 2Phenol Extraction –gentle rocking several hours 3Ethanol Precipitation 4RNAse followed by proteinase K 5Repeat Phenol Extrac- tion and EtOH ppt EtOH Precipitation 2-2.5 volumes EtOH, -20 o high salt, pH 5-5.5 centrifuge or ‘spool’ out 3Dr.Saba Abdi

4. Isolation of RNA Special Considerations RNAse inhibitors! extraction in guanidine salts phenol extractions at pH 5-6 (pH 8 for DNA) treatment with RNase-free DNase selective precipitation of high MW forms (rRNA, mRNA) with LiCl oligo-dT column 4Dr.Saba Abdi

5. Plasmid Miniprep Protocol 1. Solubilize bacteria in alkali solution 2. Neutralize with Na-acetate 3. Centrifuge, discard pellet 4. Mix supernatant with resin + chaotropic agent 5. Wash resin 6. Elute DNA with low salt buffer Adsorption Methods nucleic acids selectively absorb to silica or resins in the presence of certain chaotropic agents or salts applications: plasmid preps fragments after electrophoresis PCR templates 5Dr.Saba Abdi

6. Density Gradient Centrifugation rate zonal/sucrose (size fractionation) electrophoresis more common isopycnic/CsCl (density) DNA ~1.7 g/cm 3 protein ~1.3 g/cm 3 RNA > DNA ssDNA > dsDNA GC content 20 40 6080 % GC base pairs 1.68 1.70 1.72 1.74 density (g/cm 3 ) 6Dr.Saba Abdi

7. CsCl Gradients Applications large scale preparations high purity ‘satellite’ DNA RNA ‘cushions’ CsCl Gradients 7Dr.Saba Abdi

8. Using Spectroscopy to analyze DNA DNA absorbs UV light with a major peak at 260 nm Optical Density Wave Length 260 This absorption is useful because it varies with the structure of DNA (&RNA) i.e. extinction coefficient depends on the structure dsDNA Low extinction coefficient ssDNA Higher extinction coefficient 8Dr.Saba Abdi

9. Evaluation of Nucleic Acids spectrophotometrically quantity quality fluorescent dyes gel electrophoresis 9Dr.Saba Abdi

10. Agarose Gel Stained with ethidium bromide (EtBR) to Visualize the DNA Agarose Gel Stained with ethidium bromide (EtBR) to Visualize the DNA Screening PCR products to test for the presence of specific DNA sequences 500 bp molecular weight markers molecular weight markers correct PCR product 600 bp 700 bp 1000 bp slots where DNA is loaded 10Dr.Saba Abdi

11. Intercalating Agents Distort the Double Helix Several hydrophobic molecules containing flat aromatic and fused heterocyclic rings can insert between the stacked base pairs of DNA. These molecules are called intercalating agents. Intercalating agents are potential Cancer-inducing reagents. 11Dr.Saba Abdi

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13. DNA Sequencing Two Methods: chemical cleavage xxx (Maxam and Gilbert) synthetic oligonucleotides GC-rich DNA dideoxy (Sanger) based on 2’3’-dideoxynucleotides as chain terminators  H 13Dr.Saba Abdi

14. Dideoxy Chain Termination 14Dr.Saba Abdi

15. DNA sequencing: the Sanger (dideoxy) method Figure 7-29b,c 15Dr.Saba Abdi

16. NTP, dNTPs and ddNTPs 16Dr.Saba Abdi

17. DNA sequencing: the Sanger method Figure 7-29a Four separate polymerization reactions are performed 17Dr.Saba Abdi

18. DNA Sequencing 18Dr.Saba Abdi

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20. CGGGCGTCGGGCGT Sequence 5’ to 3’ Reading a DNA Sequencing Gel 20Dr.Saba Abdi

21. Semi-Automated Sequencing  thermal cycler fluorescent ddNTPs unique spectra measure intensity of DNA products on gel 21Dr.Saba Abdi

22. Automated DNA Sequencing with Fluorescent Dyes Each different ddNTP is coupled to a different colored fluorescent dye ddTTP is red; ddGTP is black etc. 22Dr.Saba Abdi