2. Introduction
DNA (Deoxyribo Nucleic Acid) is a long stringy molecule that can be extracted from any
biological material suchaslivingorconservedtissues,cellsandvirusparticles.
Thefirstisolationof DNAwasdonein1869byFriedrichMiescher
.
The extraction of DNA is pivotal to biotechnology. It is the starting point for numerous
applications, rangingfromfundamental researchtoroutinediagnosticandtherapeuticdecision-
making. Extractionandpurificationarealso essential to determiningtheuniquecharacteristics
of DNA, includingitssize,shapeandfunction.
The approach to preparation of DNA from plants is determined by the species, the type of
tissueorsampleavailableandtheanalysisrequiredontheDNA.
5. Extractionof DNA fromplantmaterial
Isolatespecific
tissues
Components of the process may
include isolation of specific tissue,
grinding (or other mechanical
disruption), extraction into solution,
Removal of
contaminants
Removal of RNA
contamination
solvent purification and
precipitation.
Precipitation
7. Cell Lysisor Cell distruption:
Cell disruption is a method or process for releasing
biological moleculesfrominsideacell .
Vortexing
Sonication
Physical
Manual Grinding
Cell Lysis
Treatwithlysozyme,
EDTA, SDS
Chemical
11. Phenol–chloroformextraction
A phenol-chloroformextractionisaliquid-liquidextraction.
Basedonthedifferential solubilitiesof theindividualmolecules
In 1953, Grassmann & Defner described the efficacy of phenol at
extracting proteins from aqueous solution . Utilizing this find,
Kirby demonstratedtheuseof phenol toseparatenucleicacidsfrom
proteinsin1956.
Bestmethodsof DNA extraction.
The method is also called as a phenol-chloroform and isoamyl
alcohol,PCI methodof DNA extraction.
12. Cont...
Aqueous samples are mixed with equal
volumesof aphenol:chloroformmixture.
After centrifugation, two distinct phases
areformed.
The phenol:chloroform mixture is
immisciblewithwater.
Theaqueousphaseisontopbecauseitis
less dense than the organic phase
(phenol:chloroform).
The proteins and hydrophobic lipids will
partition into the lower organic phase
while the nucleic acids (as well as other
contaminants such as salts, sugars, etc.)
remainintheupperaqueousphase.
14. Cont...
ThesampleisincubatedwithproteinaseK for2hours.Thiswill
digestall theproteinpresentinsidethesample.
ImmediatelyaftertheproteinaseK digestion,thesampleis
precipitatedbychilledalcohol.
By centrifugingsample,all othercell debrisareremoved.
Finally,theDNA pelletisdissolvedinTEbuffer.
Thismethodof DNA extractionisrapidandeasy.Eventheyieldisveryhigh.However,
thequalityof DNA isamajorconcernforthismethod.
15. Saltingout
• This method is used to isolate the DNA using the different optimal
concentrations of various salts like Tris-HCl, KCl, MgCl2, NaCl used as the
buffer.
• AsafirststepinseparatingtheproteinsfromtheDNA, thehighconcentrations
of NaCl saltisadded. TheNa+ionsneutralizethenegativechargeontheDNA,
whereastheCl- ionsneutralizethepositivechargesontheproteins.
• With their charges neutralized, the proteins and DNA no longer form strong
ionicbondstooneanotherandthereforecanbeeasilyseparated.
• Therequirementof deproteinizingcell digestswithhazardousorganicsolvents
likephenol, chloroformandisoamylalcohol. Thisisachievedby saltingoutthe
cellular proteins by dehydration and precipitation with a saturated sodium
chloridesolution.
18. Magneticbeads
MagneticparticlesusedformagneticDNApurification
Magneticparticlesmadefromsyntheticpolymers, porousglass, ormetallicmaterials
likeiron-oxide.
Theparticlescanbecoatedwithfunctional groupsorcanbeleftuncoated. Whilecoated
particlesboundwithcarboxylicacidarethemostefficientatbindingDNA.
The development of high yield magnetic particles that don't require a coating are
desiredbecausethelack of acoatingandfunctional groupsallowsahighersurfacearea
forbindingnucleicacid.
Magnetic DNA purification is a clear improvement upon centrifuge-dependent
isolationtechniqueswhensemi-automaticorfully automaticsystemsareconsidered.
20. FTA Paper
Developed by Burgoyne and Fowler at Flinders
University inAustraliainthe1980.
For protecting nucleic acid samples from degradation by
nucleasesandotherprocesses.
A sample containing DNA applied to the treated filter
paperforpreservationandlongtermstorage.
A marketable advantage of the FTA® technology is that
samples spotted on treated cards may be stored at room
temperature. Thechemicals ontheFTA cards enhancethe
preservation of the DNA and inactivate many dangerous
pathogens that may be found in liquid blood samples or
driedbiological stains. Becausethecardsaresmall insize
(approximately 3.5” x 5”), they are easily packaged,
shipped,andstoredfordatabasing.
22. Estimationof thequantityof DNA
• Theconcentrationof DNA inanextractcanbemeasuredin
several ways.
• TheA of thepreparationisagoodmeasureof concentration
260
if theDNA isrelativelypure(A = 1 for 50 pg ml−1 DNA).
260
• Impurepreparationscontainothermaterial absorbinginthe
UV range.
• Gel electrophoresisonagaroseallowsestimationof both
qualityandquantity.FluorometicmethodsforDNA estimation
arealsoavailable.Thesearelikelytobemoreaccuratethan
UV-basedmethods,especiallyforimpuresamples.
23. Estimationof thequalityof DNA
• Thepurityof DNA canbeestimatedfromtheUV
absorbance(A260/A280=1.8forpureDNA).
• Electrophoresiscanbeusedtoassessthesizeof DNA
moleculesinextracts.Digestionwithrestriction
enzymesisoftenusedtotestforDNA purityand
suitabilityforuseinrecombinantDNA protocols.
24. AgaroseGel Electrophoresis
Thismethodof quantificationisbasedontheethidiumbromidefluorescentstaining
of DNA. Ethidium bromide is a fluorescent dye, which intercalates between the
stacked bases. The fluorescent yield of the dye : DNA complex is much greater
thantheunbounddye.
UV irradiation at 254nm is absorbed by the DNA and transmitted to the dye and
the bound dye itself absorbs radiation at 302nm and 366nm. This energy is
retransmittedat590nm.
The quantity of DNA can be estimated by comparing the fluorescent yield of the
sampleswithaseriesof standards.
This provides a very rapid and sensitive means of estimating the nucleic acid
concentration. A large number of samples with as little as 5ng of DNA can be
quantified. Besides quantification, it also allows provides the advantage of
analyzingthequalityof theDNA
25. UV spectroscopy
Analysis of UV absorption by the nucleotides provides a simple and accurate estimation of
theconcentration of nucleic acids in a sample. Purines and pyrmidines in nucleic acid show
absorptionmaximaaround260nm(eg., dATP:259nm; dCTP:272nm; dTTP: 247nm)
Theratioof OD /OD shouldbedeterminedtoassessthepurity of thesample.
260 280
Theamountof DNAcanbequantifiedusingtheformula:
DNA concentration(µg/ml) = OD260x 100(dilutionfactor) x 50µg/ml
1000
Inferences:
A ratiobetween1.8-2.0denotesthattheabsorptionintheUV rangeisduetonucleicacids.
A ratiolowerthan1.8indicatesthepresenceof proteinsand/orotherUV absorbers.
A ratio higher than 2.0 indicates that the samples may be contaminated with chloroform or
phenol. Ineithercase(<1.8or>2.0)itisadvisabletore-precipitatetheDNA.
