chapter 1 nucleic acid extraction

8/3/2019 Chapter 1 Nucleic Acid Extraction

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By K.H.Timotius

Krida Wacana Christian University(UKRIDA) Jakarta Indonesia

Lecture 1. Nucleic acid extraction(DNA and RNA)


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Definition Nucleic acid extraction

is the isolation and

purification of DNA(deoxyribonucleicacid) or RNA(ribonucleic acid)


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Outline

1. DNA Collection, sample, and storage

2. Extraction

3. Assessment of quality and quantity

4. Nucleic acid storage

5. Electrophoresis


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1. DNA collection, sample and storage

Whole blood

Bone marrow

Serum/plasma Buccal cells

Cultured cells

Blood spots

Body fluids

Bronchial lavage

Amniotic

Semen

Urine

Tissue samples

Fresh/frozenParaffin-embedded

Hair (shaft/root)


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1. DNA collection, sample andstorage (cont.)

Sample

Two types of tissue: fresh andpreserved.

The damaging action of tissueendonucleases.

Endonuclease: DNase and Rnase


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1. DNA collection, sample andstorage (cont.)

Temperature storage for DNA

Purified DNA may be refrigerated at4oC for up to 3 years

Samples kept over 3 years should befrozen at -70oC


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Specimen Storage RequirementsDNABlood, Bone Marrow, Other Fluids

2225 C Not recommended (1 year.


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Specimen StorageRequirements RNABlood, Bone Marrow, Other Fluids

2225 C Not recommended within 2 hours 28 C Not recommended within 2 hours

20 C Not recommended 24 weeks NOTE: Do not freeze blood or bone marrow before

lysing red blood cells (RBCs).70 C Preferred storage condition

NOTE: Do not freeze blood or bone marrow beforelysing red blood cells (RBCs)


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Blood and bone marrow Collection tubes are EDTA or ACD

5 15 ml

Sample should not be frozen for transport

4 25o

CNotes:

EDTA: Ethylenediaminetetraacetic acid

ACD: Acid-Citric-Dextrose


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Ethylenediaminetetraacetic acid


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EDTA EDTA is used extensively in the analysis of blood.

It is an anticoagulant for blood samples.

In biochemistry and molecular biology, ion

depletion is commonly used to deactivate metal-dependent enzymes, either as an assay for theirreactivity or to suppress damage to DNA orproteins.
http://en.wikipedia.org/wiki/Anticoagulanthttp://en.wikipedia.org/wiki/Biochemistryhttp://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Metalloenzymehttp://en.wikipedia.org/wiki/Metalloenzymehttp://en.wikipedia.org/wiki/Metalloenzymehttp://en.wikipedia.org/wiki/Metalloenzymehttp://en.wikipedia.org/wiki/Metalloenzymehttp://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Biochemistryhttp://en.wikipedia.org/wiki/Anticoagulant


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ACD Acid Citrate Dextrose Solution (sometimes

called Anticoagulant Citrate Dextrose Solution) isa solution of citric acid, sodium citrate anddextrose in water.

It is mainly used as an anticoagulant to preserveblood specimens required for tissue typing, it isalso used during procedures such asplasmapheresis instead of heparin.

Two different solutions (Solution A and B) aredefined by the United States Pharmacopeia.
http://en.wikipedia.org/wiki/Citric_acidhttp://en.wikipedia.org/wiki/Monosodium_citratehttp://en.wikipedia.org/wiki/Dextrosehttp://en.wikipedia.org/wiki/Anticoagulanthttp://en.wikipedia.org/wiki/Tissue_typinghttp://en.wikipedia.org/wiki/Plasmapheresishttp://en.wikipedia.org/wiki/Heparinhttp://en.wikipedia.org/wiki/United_States_Pharmacopeiahttp://en.wikipedia.org/wiki/United_States_Pharmacopeiahttp://en.wikipedia.org/wiki/Heparinhttp://en.wikipedia.org/wiki/Plasmapheresishttp://en.wikipedia.org/wiki/Tissue_typinghttp://en.wikipedia.org/wiki/Anticoagulanthttp://en.wikipedia.org/wiki/Dextrosehttp://en.wikipedia.org/wiki/Monosodium_citratehttp://en.wikipedia.org/wiki/Citric_acid


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Chaotropic agent A denaturating agent is a substance which disrupts thethree dimensional structure in macromolecules such as

proteins, DNA, or RNA and denatures them.

A denaturating agent is a chaotropic agent, but chaotropicagents aren't necessarily denaturating agents.

Chaotropic agents disrupt the intermolecular forcesbetween water molecules, allowing proteins and othermacromolecules to dissolve more easily.

Chaotropic agents interfere with stabilizing intramolecularinteractions mediated by non-covalent forces such ashydrogen bonds, van der Waals forces, and hydrophobic

effects. Chaotropic reagents include: Urea 6 - 8 mol/l; Thiourea 2

mol/l; Guanidinium chloride 6 mol/l; Lithium perchlorate4.5 mol/l
http://en.wikipedia.org/wiki/Macromoleculehttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/RNAhttp://en.wikipedia.org/wiki/Denaturation_(biochemistry)http://en.wikipedia.org/wiki/Covalenthttp://en.wikipedia.org/wiki/Hydrogen_bondhttp://en.wikipedia.org/wiki/Van_der_Waals_forceshttp://en.wikipedia.org/wiki/Hydrophobic_effecthttp://en.wikipedia.org/wiki/Hydrophobic_effecthttp://en.wikipedia.org/wiki/Ureahttp://en.wikipedia.org/wiki/Molarityhttp://en.wikipedia.org/wiki/Thioureahttp://en.wikipedia.org/wiki/Molarityhttp://en.wikipedia.org/wiki/Guanidinium_chloridehttp://en.wikipedia.org/wiki/Lithium_perchloratehttp://en.wikipedia.org/wiki/Lithium_perchloratehttp://en.wikipedia.org/wiki/Lithium_perchloratehttp://en.wikipedia.org/wiki/Guanidinium_chloridehttp://en.wikipedia.org/wiki/Guanidinium_chloridehttp://en.wikipedia.org/wiki/Guanidinium_chloridehttp://en.wikipedia.org/wiki/Molarityhttp://en.wikipedia.org/wiki/Thioureahttp://en.wikipedia.org/wiki/Molarityhttp://en.wikipedia.org/wiki/Ureahttp://en.wikipedia.org/wiki/Hydrophobic_effecthttp://en.wikipedia.org/wiki/Hydrophobic_effecthttp://en.wikipedia.org/wiki/Van_der_Waals_forceshttp://en.wikipedia.org/wiki/Hydrogen_bondhttp://en.wikipedia.org/wiki/Covalenthttp://en.wikipedia.org/wiki/Denaturation_(biochemistry)http://en.wikipedia.org/wiki/RNAhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Macromolecule


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GITC (guanidium isothyocyanate)

a general protein denaturant, being achaotropic agent,
http://en.wikipedia.org/wiki/Chaotropic_agenthttp://en.wikipedia.org/wiki/Chaotropic_agenthttp://en.wikipedia.org/wiki/Chaotropic_agenthttp://en.wikipedia.org/wiki/Chaotropic_agent


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Serum

Collection tubes with no additives

100 l 1 ml

Transported at 20 25o

C


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Urine

Urine container should be used forcollection

At least 1 ml should be collected

Transported at 4 25oC


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2. ExtractionChemical treatments cause cellsand nuclei to burst

The nucleic acid is inherentlysticky, and can be pulled out ofthe mixture

This is called spooling nucleicacid


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Spooled NA
http://carnegieinstitution.org/first_light_case/horn/DNA/images/dnaglopp.jpg


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Extraction

Tissue isolation, membranedisruptionand cell lysis

Paraffin-embedded tissue require

deparaffinsation (heating or solvents likexylene)

Blood samples

Organic extraction

Inorganic extraction

RNA extraction


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Paraffin-embedded Tissue Sections

Genetic testing, infectious diseasetesting, identity testing

Formalin-fixed tissue is suitable.

Mercury or other heavy metalfixatives are not acceptable.

Tissue sections on glass slides canbe used for in situ applications andmicrodissection techniques.


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Blood samples WBCs RBCs

Plasma/serum


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Separate WBCs from RBCs, if necessary

Lyse WBCs or other nucleated cells

Denature/digest proteins

Separate contaminants (e.g., proteins,heme)

from DNA

Precipitate DNA if necessary

Resuspend DNA in final buffer

Basic Steps in IsolatingDNA from Clinical Specimens


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Membrane disruption/ lysis

Detergent SDS: sodium dodecylsulfate

Proteolytic agents: Proteinase K


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Organic extraction

Phenol-chloroform extraction

Separation of protein into organicphase and nucleic acid intoaqueous phase.

Phenol pH: 7.8 = 8.0 which prevent

nucleic acid from remaining in theorganic phase.


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High MW Genomic DNA Isolation

Typical Procedure1 Cell Lysis

0.5% SDS + proteinaseK (55o several hours)

2 Phenol Extraction

gentle rocking severalhours

3 Ethanol Precipitation

4 RNAse followed by proteinase K

5 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 Procedure

1 Cell Lysis: 0.5% SDS +

proteinase K (55o severalhours)

2 Phenol Extraction: gentlerocking several hours

3 Ethanol Precipitation

4 RNAse followed byproteinase K

5 Repeat Phenol Extractionand EtOH ppt

EtOH Precipitation 2-2.5 volumes EtOH, -20o high salt, pH 5-5.5 centrifuge or spool out

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SDS Sodium dodecyl sulfate (SDS or NaDS),

sodium laurilsulfate or sodium lauryl sulfate(SLS) is an organic compound with the formulaCH3(CH2)11OSO3Na).

It is an anionicsurfactant used in many cleaningand hygiene products. The salt is of anorganosulfate consisting of a 12-carbon tailattached to a sulfate group, giving the materialthe amphiphilic properties required of a detergent.

Being derived from inexpensive coconut andpalm oils, it is a common component of manydomestic cleaning products.
http://en.wikipedia.org/wiki/Sulfatehttp://en.wikipedia.org/wiki/Sulfatehttp://en.wikipedia.org/wiki/Sodiumhttp://en.wikipedia.org/wiki/Anionhttp://en.wikipedia.org/wiki/Surfactanthttp://en.wikipedia.org/wiki/Organosulfatehttp://en.wikipedia.org/wiki/Sulfatehttp://en.wikipedia.org/wiki/Amphiphilichttp://en.wikipedia.org/wiki/Detergenthttp://en.wikipedia.org/wiki/Detergenthttp://en.wikipedia.org/wiki/Amphiphilichttp://en.wikipedia.org/wiki/Sulfatehttp://en.wikipedia.org/wiki/Organosulfatehttp://en.wikipedia.org/wiki/Surfactanthttp://en.wikipedia.org/wiki/Anionhttp://en.wikipedia.org/wiki/Sodiumhttp://en.wikipedia.org/wiki/Sulfatehttp://en.wikipedia.org/wiki/Sulfate


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SDS


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cell growthcell harvest andlysis

DNA purification

DNA purification: overview

DNA concentration


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Bacterial genomic DNA prep: cell extract

Lysis:

Detergents Organic solvent Proteases (lysozyme) Heat

cell extract


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Genomic DNA prep: removing proteins and RNA

Add the enzyme RNase to degrade RNA in the aqueous

layer

Need to mix gently! (to avoid shearing breakage of thegenomic DNA)

chloroform


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2 ways to concentrate the genomic DNA

70% final conc.

spooling Ethanol precipitation


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Plasmids: vehicles of recombinant DNA

Bacterial cell

genomic DNA plasmids

Non-chromosomal DNAReplication: independent of the chromosomeMany copies per cellEasy to isolateEasy to manipulate

Plasmid purification: alkaline lysis


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Plasmid purification: alkaline lysis

Alkaline

conditionsdenatureDNA

Neutralize:

genomic DNAcant renature(plasmidsCAN because

they neverfullyseparate)


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DNA purification: phenol/chloroform extraction

1:1 phenol : chloroformor

25:24:1 phenol : chloroform : isoamyl alcohol

Phenol: denatures proteins, precipitates form atinterface between aqueous and organic layer

Chloroform: increases density of organic layer

Isoamyl alcohol: prevents foaming

Ph l t ti


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1. Aqueous volume (at least 200 microliters)

2. Add 2 volumes of phenol:chloroform, mix well

3. Spin in centrifuge, move aqueous phase to a new tube

4. Repeat steps 2 and 3 until there is no precipitate at phase interface

5. (extract aqueous layer with 2 volumes of chloroform)

Phenol extraction

Eth l i it ti (DNA t ti )


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Ethanol depletes the hydration shell surrounding DNA

Allowing cations to interact with the DNA phosphates

Reducing repulsive forces between DNA strands

Causing aggregation and precipitation of DNA

Aqueous volume (example: 200 microliters)

-- add 22 microliters sodium acetate 3M pH 5.2

-- add 1 microliter of glycogen (gives a visible pellet)

-- add 2 volumes (446 microliters) 100% ethanol

-- mix well, centrifuge at high speed, decant liquid

-- wash pellet (70% ethanol), dry pellet, dissolve in appropriate volume (then

determine DNA concentration)

Ethanol precipitation (DNA concentration)

f


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cell growthcell harvest andlysis

DNA purification

DNA purification: overview

DNA concentration


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Inorganic extraction

Salt precipitation adsorption to silica surfaces, and

anion - exchange chromatography.


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Plasmid Miniprep Protocol

1. Solubilize bacteria in alkali solution2. Neutralize with Na-acetate3. Centrifuge, discard pellet4. Mix supernatant with resin +

chaotropic agent5. Wash resin

6. Elute DNA with low salt buffer

Adsorption Methods

nucleic acids selectively absorb to silica orresins in the presence of certain chaotropicagents or salts

applications: plasmid preps fragments after

electrophoresis PCR templates

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DNA purification: silica binding

Binding occurs in presence of high saltconcentration, and is disrupted by elution withwater


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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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RNA I l ti M th d


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RNA Isolation MethodsCesium Chloride Gradient

Used mainly to get clean RNA for Northern blots Homogenize cells in guanidinium isothiocyanate and

b-mercaptoethanol solution.

Add to CsCl gradient and centrifuge for 1220 hours;RNA will be at the bottom of tube.

Re-dissolve in TE/SDS buffer.

Precipitate RNA with salt and ethanol, then rehydrate.

Advantage: high quality

Disadvantages: extremely time-consuming,hazardous materials disposal issues


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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

density(g/cm3)

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Centrifuge rotors

Fixed-angle

axis of rotation

At rest

Swinging-bucket

g

Spinning g


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Differential centrifugation of atissue homogenate (I)

1000g/10 min

Decant supernatant

3000g/10 minetc.


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Density Barrier Discontinuous Continuous

Density gradient centrifugation


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How does a gradient separate

different particles?

Least dense

Most dense


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Buoyant

densitybanding

Equilibriumdensitybanding

Isopycnicbanding

1

5

2

3

4


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Discontinuous

Resolution of density gradients

ContinuousDensity Barrier

I II


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RNA extraction

RNA extraction demands extra care. Mostforms of RNA are labile. Contaminant RNase: pretreatment with

DEPC (diethylpyrocarbonate) a strong

RNase inhibitor. Glassware can be baked at 150oC for 4

hours Plastic materials can be soaked in 0.5 M

NaOH for 10 min. Autoclave treatment for glassware and

plastic materials.

The problem(s) with RNA:


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p ( )

RNA is chemically unstable -- spontaneous cleavage ofphosphodiester backbone via intramoleculartransesterification

RNA is susceptible to nearly ubiquitous RNA-degrading

enzymes (RNases)RNases are released upon cell lysisRNases are present on the skinRNases are very difficult to inactivate

-- disulfide bridges conferring stability-- no requirement for divalent cations for

activity


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Top 10 sources of RNAse contamination(Ambion Scientific website)

1) Ungloved hands2) Tips and tubes3) Water and buffers4) Lab surfaces5) Endogenous cellular RNAses6) RNA samples

7) Plasmid preps8) RNA storage (slow action of small amounts of RNAse9) Chemical nucleases (Mg++, Ca++ at 80C for 5 +)10) Enzyme preparations


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DEPC: diethylpyrocarbonate

RNA Isolation Methods


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RNA Isolation MethodsGuanidinium-based Organic Isolation

Phenol/guanidinium solution disrupts cells,solubilizes cell components, but maintainsintegrity of RNA.

Add chloroform, mix, and centrifuge.

Proteins/DNA remain at interface. RNA is removed with aqueous top layer. RNA is precipitated with alcohol and

rehydrated.

Advantage: faster than CsCl method Disadvantages: fume hood required,

hazardous waste disposal issues

RNA Isolation Methods


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RNA Isolation MethodsNonorganic Salt Precipitation

Cell membranes are lysed and proteins aredenatured by detergent (such as SDS) in thepresence of EDTA or other RNase inhibitors.

Proteins/DNA are precipitated with a high

concentration salt solution. RNA is precipitated with alcohol and

rehydrated.

Advantages: Fast and easy, nontoxic Produces high quality RNA


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3. Assessment of quality and quantity

Maximal absorption of nucleic acid is at wavelength269 nm.

Proteins absorb well at 280 nm.

OD260 of 1.0 corresponds to approx 50 g/ml ofdouble-stranded DNA or 40 g/ml for single-strandedDNA or RNA.

OD 260/280 ratio provides an estimate of nucleic acidpurity, with a pure preparation having a ratio between 1.8and 2.0.

Dyes that bind nucleic acid are acridine orange,daminoibenzoic acid (DABA), propidium iodide, andethydium bromide.

Double-stranded and single-stranded DNA differ in their


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dsDNA

ssDNA

nucleotidesdA

dC

dG

dU

The conjugated p-electron systems ofthe purine & pyrimidine bases absorbstrongly in the UV.

The absorbance of double-strandedDNA (dsDNA) at 260 nm is less thanthat of either single-stranded DNA(ssDNA) or the free bases. This is

called hypochromism.

Double stranded and single stranded DNA differ in theiroptical absorption at 260 nm


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Using Spectroscopy to analyze DNA

DNA absorbs UV light with a major peak at 260 nm

OpticalDensity

Wave Length

This absorption is useful because itvaries with the structure of DNA(&RNA)

i.e. extinction coefficient depends on

the structure

dsDNA

Low extinctioncoefficient

ssDNA

Higher extinctioncoefficient

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Resuspending Final Nucleic Acid


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Resuspending Final Nucleic AcidSamples

Have some idea of expected nucleic acid yield. Choose diluent volume according to desired

concentration.

Calculating Expected DNA Yield

Example:1 X 107 cells X 6 pg DNA/cell X 80% yield= 48 mg DNA

Resuspend DNA in TE buffer or ultra pureDNAse-free water.

Resuspend RNA in ultra pure RNase-free water.


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Quantity from UV Spectrophotometry

DNA and RNA absorb maximally at260 nm.

Proteins absorb at 280 nm.

Background scatter absorbs at 320nm.


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[DNA] =(A260 A320) X dilution factor X 50 g/mL

[RNA] =

(A260 A320) X dilution factor X 40 g/mL

Concentration = g of DNA or RNA per mL ofhydrating solution


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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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Multiply the concentration of theDNA or RNA sample by the

volume of hydrating solution added.Example for DNA: 150 g/mL X 0.1 mL = 15 g

Concentration fromUV Spec. (g DNAper ml of hydrating

solution)

Volume ofhydrationsolution

DNA yield

Quantity from UV SpectrophotometryCalculating Yield


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A260/A280 = measure of purity

(A260 A320)/(A280 A320)

1.7 2.0 = good DNA or RNA


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4. Nucleic acid storage

To prevent enzymatic or physical damage to thepurified product.

Chelating agents, chaotrophic agents,refrigeration.

DNA can be stored for long periods in aTRIS=EDTA buffer at 4oC.

RNA, more labile, should be stored at -80oC insimilar buffer.

DNA and RNA can be stored as an ethanolprecipitate, with -20oC being the optimal storagetemperature.


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5. Electrophoresis

DNA can be separated based onsize and charge

The phosphate groups are

negatively charged

DNA is placed in a gel andelectricity is run through

Separates DNA (or RNA or Protein) fragments on


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the basis of charge and size

Because DNA is an acid, it looses protons in basic

buffers; thus it has a negative charge that is uniformper unit length

Agarose (a polysaccharide) or other gel matricesare difficult for large DNA fragments to move

through The larger the fragment, the more difficulty it has

moving through gels

By placing DNA in a gel, then applying a voltage

across the gel, the negatively charged DNA willmove toward the anode (positive electrode)

Large fragments lag behind while small fragmentsmove through the gel relatively rapidly


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Agarose

Agar consists of a mixture ofagarose and agaropectin.

The predominant componentagarose is a linear polymer,made up of the repeatingmonomeric unit ofagarobiose.

Agarobiose is a disaccharidemade up of D-galactose and3,6-anhydro-L-galactopyranose.

Agaropectin is aheterogeneous mixture ofsmaller acidic molecules thatgel poorly.


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Electrophoresis

Negative DNA moves toward thepositive end

Smaller fragments move farther andfaster

El t h i


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75

Electrophoresis


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What is the electrical charge of


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What is the electrical charge ofDNA?

Negative, so DNA piecesmigrate toward the positivepole

Smaller fragments movefaster and travel fartherthan larger fragments.

Fragments of different sizes

appear as bands on the gel

Agarose GelStained with ethidium bromide (EtBR) to Visualize the DNA


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Stained with ethidium bromide (EtBR) to Visualize the DNA

Screening PCRproducts to testfor the presence

of specific DNAsequences

500 bp

molecularweight

markers

molecularweight

markers

correctPCR

product

600 bp700 bp

1000 bp

slots where

DNA is loaded

78 Dr.Saba Abdi

G l El t h i


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Gel Electrophoresis

+

-

Directionof

DNATravel

Wells

Small

Large


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DNA Si f A G l El t h i


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100 bp ladder

1 kb ladderLambda DNA cutwith Hind IIILambda DNA

48,500 bp(48.5 kb)

12,218 bp

23,130 bp

9,416 bp

6,557 bp

4,361 bp

2,322 bp

2,027bp 517 bp

1,636 bp

3,054 bp

6,018 bp

100 bp

300 bp

600 bp

1,000 bp

1,500 bp

1,018 bp

2,036 bp

DNA Size from Agarose Gel Electrophoresis:Compares unknown DNA to known size standards

DNA Quality from Agarose Gel


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Lambda DNAmarker

Human Whole Blood DNA

Lambda DNA cut withHind III marker

Whole blood genomic DNA

Q y gElectrophoresis


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Text Art Page 91 The electrophoretic mobility of a DNAfragment is inversely proportional tothe log of its size.


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Figure 5.2b10

20

30

40

50

60

70

3 mm

Agarose gel electrophoresis


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Agarose gel electrophoresis

Agarose (%) Standard NuSieve NuSieve 3:1

0.5 700 bp-25 Kbp

0.8 500 bp-15 Kbp 800 bp-10 Kbp

1.0 250 bp-12 Kbp 400 bp-8 Kbp

1.2 150 bp-6 Kbp 300 bp-7 Kbp

1.5 80 bp-4 Kbp 200 bp-4 Kbp

2.0 100 bp-3 Kbp

3.0 50 bp-1 Kbp 500 bp-1 Kbp

4.0 100 bp-500 bp

6.0 10 bp-100 bp


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Pulsed Field Gel Electrophoresis

agarose gel electrophoresis is a fundamental technique in molecularbiology but is generally unable to resolve fragments greater than 20kilobases in size (whole microbial genomes are usually greater than1000 kilobases in size)

PFGE (pulsed field gel electrophoresis) is a adaptation ofconventional agarose gel electrophoresis that allows extremelylarge DNA fragments to be resolved (up to megabase sizefragments)

essential technique for estimating the sizes of wholegenomes/chromosomes prior to sequencing and is necessary for

preparing large DNA fragments for large insert DNA cloning andanalysis of subsequent clones

also a commonly used and extremely powerful tool for genotypingand epidemiology studies for pathogenic microorganisms


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Principle of PFGE

two factors influence DNA migration rates through conventional gels

- charge differences between DNA fragments

- molecular sieve effect of DNA pores

DNA fragments normally travel through agarose pores as sphericalcoils, fragments greater than 20 kb in size form extended coils andtherefore are not subjected to the molecular sieve effect

the charge effect is countered by the proportionally increasedfriction applied to the molecules and therefore fragments greaterthan 20 kb do not resolve

PFGE works by periodically altering the electric field orientation

the large extended coil DNA fragments are forced to changeorientation and size dependent separation is re-establishedbecause the time taken for the DNA to reorient is size dependent


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Principle of PFGE

the most important factor in PFGE resolution is switching time,longer switching times generally lead to increased size of DNAfragments which can be resolved

switching times are optimised for the expected size of the DNAbeing run on the PFGE gel

switch time ramping increases the region of the gel in which DNAseparation is linear with respect to size

a number of different apparatus have been developed in order to

generate this switching in electric fields however most commonlyused in modern laboratories are FIGE (Field Inversion GelElectrophoresis) and CHEF (Contour-Clamped HomogenousElectrophoresis)

CHEF


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+

+

+

+ +

+

+

+

--

-

- -

-

-

-

Electric Field 1 Electric Field 2

Switch Time

But what if you want to separate larger fragments such as entire yeast


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But, what if you want to separate larger fragments, such as entire yeastchromosomes?

Pulsed-field gel electrophoresis (PFGE) can resolve fragments from 200 Kpb(0.2 Mbp) to 6000 Kbp (6 Mbp).

+

|

+ |

+

|

+ |

+

|


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Isolation of Nucleic Acids


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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)

92 Dr.Saba Abdi


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Downstream Applications

After DNA is extracted, it is used as atemplate in further molecular techniques suchas

PCR (polymerase chain reaction) RFLP (restriction fragment length polymorphism)

Southern Blotting

What do we need DNA for?


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What do we need DNA for?

Detect, enumerate, clone genesDetect, enumerate speciesDetect/sequence specific DNA regionsCreate new DNA constructs (recombinant DNA)

What about RNA?

Which genes are being transcribed?When/where are genes being transcribed?What is the level of transcription?

DEPC: diethylpyrocarbonateDEPC: diethylpyrocarbonate

chapter 1 nucleic acid extraction

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