Chapter 1 Nucleic Acid Extraction
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Transcript of 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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Quantity from UV Spectrophotometry
[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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Quantity from UV Spectrophotometry
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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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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
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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
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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