DNA isolation .ppt by dffhrtcv3

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									     Isolation of Nucleic Acids
Goals:                        Types of Methods:
• removal of proteins         • differential solubility
• DNA vs RNA                  • ‘adsorption’ methods
• isolation of a specific     • density gradient
  type of DNA (or RNA)          centrifugation
Types of DNA:
• genomic                       General Features:
  (chromosomal)                 • denaturing cell lysis (SDS,
• organellar (satellite)          alkali, boiling, chaotropic)
• plasmid (extra-               •  enzyme treatments
  chromosomal)                        - protease
• phage/viral (ds or ss)              - RNase (DNase-free)
• complementary                       - DNase (RNase-free)
  (mRNA)                 Dr.Saba Abdi                       1
     High MW Genomic DNA Isolation
Typical Procedure                 Phenol Extraction
1 Cell Lysis                      • mix sample with equal volume
   – 0.5% SDS + proteinase          of sat. phenol soln
     K (55o several hours)        • retain aqueous phase
2 Phenol Extraction               • optional chloroform/isoamyl
   – gentle rocking several         alcohol extraction(s)
3 Ethanol Precipitation
4 RNAse followed by                           aqueous phase
  proteinase K                                 (nucleic acids)
5 Repeat phenol extrac-                       phenol phase
  tion and EtOH ppt                            (proteins)
                              Dr.Saba Abdi                       2
     High MW Genomic DNA Isolation
Typical Procedure                      EtOH Precipitation
1 Cell Lysis                           • 2-2.5 volumes EtOH, -20o
   – 0.5% SDS + proteinase             • high salt, pH 5-5.5
     K (55o several hours)             • centrifuge or ‘spool’ out
2 Phenol Extraction
   – gentle rocking several
3 Ethanol Precipitation
4 RNAse followed by
  proteinase K
5 Repeat Phenol Extrac-
  tion and EtOH ppt
                              Dr.Saba Abdi                     3
    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             4
         Adsorption Methods
• nucleic acids selectively absorb to silica or
  resins in the presence of certain chaotropic
  agents or salts
                        Plasmid Miniprep Protocol
• applications:
  • plasmid preps        1. Solubilize bacteria in alkali
  • fragments after           solution
    electrophoresis      2. Neutralize with Na-acetate
  • PCR templates        3. Centrifuge, discard pellet
                         4. Mix supernatant with resin
                              + chaotropic agent
                         5. Wash resin
                         6. Elute DNA with low salt
                      Dr.Saba Abdi                    5
 Density Gradient Centrifugation
• rate zonal/sucrose (size fractionation)
  • electrophoresis more common

• isopycnic/CsCl (density)
  •   DNA ~1.7 g/cm3
  •   protein ~1.3 g/cm3                   1.74

                             density (g/cm3)
  •   RNA > DNA
  •   ssDNA > dsDNA                        1.72
  •   GC content


                                                      20      40    60       80
                           Dr.Saba Abdi                    % GC base pairs   6
                       CsCl Gradients
                  • large scale preparations
                  • high purity
                  • ‘satellite’ DNA
                  • RNA ‘cushions’

CsCl Gradients
                 Dr.Saba Abdi             7
                     Using Spectroscopy to analyze DNA

                  DNA absorbs UV light with a major peak at 260 nm
Optical Density

                                              This absorption is useful
                                              because it varies with the
                                              structure of DNA (&RNA)
                                              i.e. extinction coefficient
                          Wave Length         depends on the structure

                             dsDNA                               ssDNA
                             Low extinction                      Higher extinction
                             coefficient                         coefficient
                                          Dr.Saba Abdi                           8
Evaluation of Nucleic Acids
    • spectrophotometrically
      • quantity
      • quality
    • fluorescent dyes
      • gel electrophoresis

       A260          1.0  50 g/ml
       A260/A280     1.6 - 1.8
       A260          1.0  40 g/ml
       A260/A280     ~2.0

               Dr.Saba Abdi           9
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

       molecular            correct       molecular
        weight               PCR           weight
       markers              product
                         Dr.Saba Abdi     markers          10
          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
                          Dr.Saba Abdi                11
Dr.Saba Abdi   12
           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

                       Dr.Saba Abdi          13
Dideoxy Chain Termination

              Dr.Saba Abdi   14
DNA sequencing: the Sanger (dideoxy)

 Figure 7-29b,c   Dr.Saba Abdi    15
NTP, dNTPs and ddNTPs

       Dr.Saba Abdi     16
   DNA sequencing: the Sanger method

Four separate polymerization
reactions are performed

                                              Figure 7-29a
                               Dr.Saba Abdi         17
DNA Sequencing

                 Dr.Saba Abdi   18
Dr.Saba Abdi   19
Reading a DNA Sequencing Gel

                             Sequence 5’ to 3’
          Dr.Saba Abdi                     20
Semi-Automated Sequencing
• thermal cycler
• fluorescent ddNTPs
   • unique spectra
• measure intensity of
  DNA products on gel

                Dr.Saba Abdi   21
Automated DNA Sequencing with Fluorescent Dyes

 Each different ddNTP is coupled to a different colored fluorescent dye
                              is black etc.
         ddTTP is red; ddGTP Dr.Saba Abdi                             22

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