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 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) [Link] Abdi 1
High MW Genomic DNA Isolation
Typical Procedure
1 Cell Lysis
0.5% SDS + proteinase K (55o several hours)
Phenol Extraction
mix sample with equal volume of sat. phenol soln retain aqueous phase optional chloroform/isoamyl alcohol extraction(s)
2 Phenol Extraction
gentle rocking several hours
3 Ethanol Precipitation 4 RNAse followed by proteinase K 5 Repeat phenol extraction and EtOH ppt
[Link] Abdi
aqueous phase (nucleic acids) phenol phase (proteins)
2
High MW Genomic DNA Isolation
Typical Procedure
1 Cell Lysis
0.5% SDS + proteinase K (55o several hours)
EtOH Precipitation
2-2.5 volumes EtOH, -20o high salt, pH 5-5.5 centrifuge or spool out
2 Phenol Extraction
gentle rocking several hours
3 Ethanol Precipitation 4 RNAse followed by proteinase K 5 Repeat Phenol Extraction and EtOH ppt
[Link] 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
[Link] Abdi
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 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 [Link] Abdi 5 buffer
Density Gradient Centrifugation
rate zonal/sucrose (size fractionation)
electrophoresis more common
isopycnic/CsCl (density)
density (g/cm3)
DNA ~1.7 g/cm3 protein ~1.3 g/cm3 RNA > DNA ssDNA > dsDNA GC content
1.74 1.72 1.70 1.68 20
[Link] Abdi
% GC base pairs
40
60
80
6
CsCl Gradients
Applications large scale preparations high purity satellite DNA RNA cushions
CsCl Gradients
[Link] Abdi
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)
Wave Length
dsDNA Low extinction coefficient
[Link] Abdi
260
i.e. extinction coefficient depends on the structure
ssDNA Higher extinction coefficient
8
Evaluation of Nucleic Acids
spectrophotometrically
quantity quality
fluorescent dyes
gel electrophoresis
A260 DNA A260/A280 A260 RNA A260/A280
1.0 50 g/ml 1.6 - 1.8 1.0 40 g/ml ~2.0
9
[Link] Abdi
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 [Link] Abdi product molecular weight 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 Cancer-inducing reagents.
[Link] Abdi 11
[Link] Abdi
12
DNA Sequencing
Two Methods:
chemical cleavage xxx (Maxam and Gilbert) synthetic oligonucleotides GC-rich DNA dideoxy (Sanger) based on 23-dideoxynucleotides as chain terminators H
[Link] Abdi 13
Dideoxy Chain Termination
[Link] Abdi
14
DNA sequencing: the Sanger (dideoxy) method
Figure 7-29b,c
[Link] Abdi
15
NTP, dNTPs and ddNTPs
[Link] Abdi
16
DNA sequencing: the Sanger method
Four separate polymerization reactions are performed
Figure 7-29a
[Link] Abdi 17
DNA Sequencing
[Link] Abdi
18
[Link] Abdi
19
Reading a DNA Sequencing Gel
Sequence 5 to 3
C G G G C G T
[Link] Abdi 20
Semi-Automated Sequencing
thermal cycler fluorescent ddNTPs unique spectra measure intensity of DNA products on gel
[Link] Abdi 21
Automated DNA Sequencing with Fluorescent Dyes
Each different ddNTP is coupled to a different colored fluorescent dye 22 ddTTP is red; ddGTP [Link] Abdietc. is black