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)

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High MW Genomic DNA Isolation

Typical Procedure1 Cell Lysis

– 0.5% SDS + proteinase K (55o several hours)

2 Phenol Extraction– gentle rocking several

hours3 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)

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High MW Genomic DNA Isolation

Typical Procedure1 Cell Lysis

– 0.5% SDS + proteinase K (55o several hours)

2 Phenol Extraction– gentle rocking several

hours3 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

dens

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 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/mlRNAA260/A280 ~2.0

• spectrophotometrically• quantity• quality

• fluorescent dyes• gel electrophoresis

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Agarose 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