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Isolation of Nucleic AcidsGoals:• 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)
1Dr.Saba Abdi
High MW Genomic DNA Isolation
Typical Procedure1 Cell Lysis
– 0.5% SDS + proteinase K (55o several hours)
2 Phenol Extraction– gentle rocking several
hours
3 Ethanol Precipitation4 RNAse followed by
proteinase K5 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)
2Dr.Saba Abdi
High MW Genomic DNA Isolation
Typical Procedure1 Cell Lysis
– 0.5% SDS + proteinase K (55o several hours)
2 Phenol Extraction– gentle rocking several
hours
3 Ethanol Precipitation4 RNAse followed by
proteinase K5 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
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Isolation of RNASpecial 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
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Plasmid Miniprep Protocol
1. Solubilize bacteria in alkali solution
2. Neutralize with Na-acetate3. Centrifuge, discard pellet4. Mix supernatant with resin
+ chaotropic agent5. Wash resin6. Elute DNA with low salt
buffer
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
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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
de
ns
ity
(g
/cm
3)
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CsCl Gradients
Applications
• large scale preparations• high purity• ‘satellite’ DNA• RNA ‘cushions’
CsCl Gradients
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Using Spectroscopy to analyze DNAUsing Spectroscopy to analyze DNA
DNA absorbs UV light with a major peak at 260 nm
Opt
ical
Den
sity
Wave Length
260
This absorption is useful because it varies with the structure of DNA (&RNA)
i.e. extinction coefficient depends on the structure
dsDNA
Low extinction coefficient
ssDNA
Higher extinction coefficient
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Evaluation of Nucleic Acids
A260 1.0 50 g/mlDNA
A260/A280 1.6 - 1.8
A260 1.0 40 g/mlRNA
A260/A280 ~2.0
• spectrophotometrically• quantity• quality
• fluorescent dyes• gel electrophoresis
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Agarose GelStained with ethidium bromide (EtBR) to Visualize the DNAAgarose GelStained with ethidium bromide (EtBR) to Visualize the DNA
Screening PCR products to test for the presence of specific DNA sequences
500 bp
molecularweightmarkers
molecularweightmarkers
correctPCR
product
600 bp700 bp
1000 bp
slotswhereDNAisloaded
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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 agentsare potential Cancer-inducing reagents.
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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
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Dideoxy Chain Termination
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DNAsequencing:theSanger(dideoxy)method
Figure 7-29b,c 15Dr.Saba Abdi
NTP, dNTPs and ddNTPs
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DNAsequencing:theSangermethod
Figure 7-29a
Four separate polymerizationreactions are performed
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DNA Sequencing
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CGGGCGT
Sequence 5’ to 3’
Reading a DNA Sequencing Gel
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Semi-Automated Sequencing
• thermal cycler• fluorescent ddNTPs•unique spectra
•measure intensity of DNA products on gel
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Automated DNA Sequencing with Fluorescent Dyes
Each different ddNTP is coupled to a different colored fluorescent dyeddTTP is red; ddGTP is black etc. 22Dr.Saba Abdi
