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)
Dr.Saba Abdi

2. 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

3. 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

4. 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

5. 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

6. 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

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

8. 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

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

10. 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

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.
Dr.Saba Abdi

12. Dr.Saba Abdi

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 ì H
Dr.Saba Abdi

14. Dideoxy Chain Termination
Dr.Saba Abdi

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

16. NTP, dNTPs and ddNTPs
Dr.Saba Abdi

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

18. DNA Sequencing
Dr.Saba Abdi

19. Dr.Saba Abdi

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

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