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

1. Disruption of cell wall and membranes to liberate cellular components.

2. Inactivation of DNA- and RNA-degrading enzymes (DNases, RNases).

3. Separation of nucleic acids from other cellular components.
         • Extraction/Precipitation method
         • Adsorption Chromatography method
Getting Prepared: Creating a Nuclease-Free Environment
 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. You should be doing this anyway for
   safety reasons, but skin cells also produce RNase7,
   a potent RNA-degrading enzyme.

 • Bake glass, metal, or ceramic equipment at high temp.
                          Cells

                                     Extract


Bacterial Cells
Or tissue culture cells
Or blood
                                  HOW?
                                  Organic
Or flies………..                     extraction


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

                an equal volume of
                organic solvent                 Centrifuge            Collect aqueous phase
               e.g. phenol, chloroform,
               or phenol:chloroform                                      Interphase

                                                                          Organic



                                              Perform additional extractions for increased purity




 Crude lysate containing                  The aqueous phase contains water-
 nucleic acids and other                  soluble molecules, including nucleic
 cell constituents                        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
     High MW Genomic DNA Isolation
                              Detail of step 4
Typical Procedure
                              EtOH Precipitation
1 Harvest cells               • 2-2.5 volumes EtOH, -20o
2 Cell Lysis                  • high salt, pH 5-5.5
   – 0.5% SDS + proteinase    • centrifuge or ‘spool’ out
     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
                        Extraction/Precipitation Method


Step 4: Nucleic Acid Precipitation

  Before     After

                            Supernatant          70% EtOH

                      Centrifuge                Wash            Centrifuge

                                Pellet

                                                                                       Dissolve pellet
                                                                                       (H2O, TE, etc.)
Add alcohol and salt to                   • Pellet down nucleic acids.
precipitate nucleic acids                 • Wash pellet with 70% ethanol to remove
from the aqueous fraction                   residual salts and other contaminants.
                                          • Discard ethanol and allow pellet to dry.
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).
    Adsorption Chromatography


Another way to selectively purify DNA


Resins (glass or chemically modified
beads) that bind nucleic acids
reversibly are packaged in columns
for easy DNA & RNA purification
               Basic Principle
        Nucleic acids within a crude lysate
          are bound to a silica surface



       The silica surface is washed with a
     solution that keeps nucleic acids bound,
        but removes all other substances



    The silica surface is washed with a solution
 unfavorable to nucleic acid binding. The solution,
containing purified DNA and/or RNA, is recovered.
                   Adsorption Chromatography Method
Step 1: Prepare crude lysate                      Step 2: Adsorb to silica surface


                                Apply to column          Centrifuge
                                                                                 Nucleic acids
                                                      Silica-gel membrane



 Extraction Buffer composition favors                                            Flow through
 DNA and RNA adsorption to silica:                                                 (discard)
 • Low pH
 • High ionic strength
 • Chaotropic salt                                         Nucleic acids bind to the membrane,
                                                           while contaminants pass through the
                                                           column.



                                                                      Having the ability to
                                                                      destabilize hydrogen
                                                                      bonding and
                                                                      hydrophobic interactions.
    Adsorption Chromatography Method
Step 3: Wash away residual contaminants

                                 Centrifuge
             Wash buffer

            Nucleic acids                     Nucleic acids




                                              Flow through
                                                (discard)
Step 4: Elute nucleic acids

                                 Centrifuge
            Elution buffer

            Nucleic acids


   Elution Buffer composition is
   unfavorable to surface binding:
   High pH
   Low ionic strength                         Nucleic acids
pH & SALT
Purifying one type of DNA
away from other DNA molecules

-Plasmids from bacterial chromosomal DNA
    Isolation of plasmid DNA
• Why plasmid?
                 Method
• Boiling lysis or alkaline lysis
• Selectively isolates the plasmid excludes
  most of the chromosomal DNA
• How is it possible to be selective
 – Plasmid DNA is almost always extracted as a
   CCC
 – Chromosomal DNA is linear fragments
 – Plasmids are much smaller than chromosomal
   DNA
             Boiling lysis

• Cell lysis with lysozyme
• Chromosomal DNA is separated from the
  plasmid DNA by boiling the lysis mixture
  followed by centrifugation
Chromosomal DNA
E. coli



          Pellet bacteria



            Lyse
                                         Boil and
                                         centrifuge




                 After centrifugation

                            Soluble
                            plasmid

                             Glutinous
                             pellet
           Alkaline lysis
The cells are lysed and their contents
 denatured with alkaline SDS. Proteins
 and high-molecular-weight chromosomal
 DNA denatured under these conditions
 precipitate leaving the supernate
 containing plasmid DNA and RNA.
SDS, alkali
alkali




  neutralize
Separation of Nucleic Acids by CeCl Gradient Centrifugation
Plasmid DNA
    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
 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
                                         1.70


                                             1.68

                                                    20      40    60       80
                                                         % GC base pairs
                              CsCl Gradients
                            Applications
                            • large scale preparations
                            • high purity
                            • RNA ‘cushions’
                            • ‘satellite’ DNA




Cesium Chloride Gradients
Assessing the Quality and Yield
       of Nucleic Acids
Evaluation of Nucleic Acids
    • spectrophotometrically
      • quantity
      • quality
    • fluorescent dyes
      • gel electrophoresis


       A260        1.0  50    mg/ml
   DNA
       A260/A280   1.6 - 1.8
       A260        1.0  40    mg/ml
   RNA
       A260/A280   ~2.0
 Nucleic Acid Analysis via UV Spectrophotometry
                                              DNA Absorption Spectra




By measuring the amount of light absorbed by your sample at specific
wavelengths, it is possible to estimate the concentration of DNA and
RNA. Nucleic acids have an absorption peak at ~260nm.
        [dsDNA] ≈ A260 x (50 µg/mL)
        [ssDNA] ≈ A260 x (33 µg/mL)
        [ssRNA] ≈ A260 x (40 µg/mL)
                          How pure is your sample?

The A260/A280 ratio is ~1.8 for dsDNA, and ~2.0 for ssRNA. Ratios lower than
1.7 usually indicate significant protein contamination.

The A260/A230 ratio of DNA and RNA should be roughly equal to its A260/A280
ratio (and therefore ≥ 1.8). Lower ratios may indicate contamination by
organic compounds (e.g. phenol, alcohol, or carbohydrates).

Turbidity can lead to erroneous readings due to light interference. Nucleic
acids do not absorb light at the 320 nm wavelength. Thus, one can correct
for the effects of turbidity by subtracting the A320 from readings at A230, A260
and A280.
Checking for Degradation: DNA

genomic
DNA
             Running your sample through an agarose
             gel is a common method for examining the
             extent of DNA degradation. Good quality
             DNA should migrate as a high molecular
             weight band, with little or no evidence of
RNA
(degraded)
             smearing.
Checking for Degradation: RNA

            Ribosomal RNA (rRNA) makes up more than
            80% of total RNA samples. Total RNA preps
            should display two prominent bands after gel
      25S   electrophoresis. These correspond to the 25S
      18S   and 18S rRNAs, which are 3.4 kb and 1.9 kb
            in Arabidopsis (respectively).

            Good quality RNA will have:
            No evidence of smearing
            25S/18S ratio between 1.8 - 2.3

				
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posted:5/17/2011
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