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) Dr.Saba Abdi

High MW Genomic DNA Isolation Typical Procedure Cell Lysis 0.5% SDS + proteinase K (55o several hours) Phenol Extraction gentle rocking several hours Ethanol Precipitation RNAse followed by proteinase K Repeat 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) Dr.Saba Abdi

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

Special Considerations 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 Dr.Saba Abdi

Adsorption Methods nucleic acids selectively absorb to silica or resins in the presence of certain chaotropic agents or salts 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 applications: plasmid preps fragments after electrophoresis PCR templates Dr.Saba Abdi

Density Gradient Centrifugation rate zonal/sucrose (size fractionation) electrophoresis more common isopycnic/CsCl (density) DNA ~1.7 g/cm3 protein ~1.3 g/cm3 RNA > DNA ssDNA > dsDNA GC content 20 40 60 80 % GC base pairs 1.68 1.70 1.72 1.74 density (g/cm3) Dr.Saba Abdi

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

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

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

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

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. Dr.Saba Abdi

Dr.Saba Abdi

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 ì H Dr.Saba Abdi

Dideoxy Chain Termination Dr.Saba Abdi

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

NTP, dNTPs and ddNTPs Dr.Saba Abdi

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

DNA Sequencing Dr.Saba Abdi

Dr.Saba Abdi

Reading a DNA Sequencing Gel T Sequence 5’ to 3’ Dr.Saba Abdi

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

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