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ISOLATION of
GENOMIC DNA
FROM
EUKARYOTIC CELLS
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DNA was isolated, analyzed and recognized as a unique
macromolecule by Friedrich Miescherin 1869
He extracted an acid-insoluble, alkali-soluble,high-phosphorus
containing substance from the nuclei of
pus cells, characterized what he recognized as a new
class of substances and named it nuclein, which we
now know as DNA.
FIRST DNA ISOLATION
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GENOMIC DNA ISOLATION FROM EUKARYOTIC CELLS
Most easily available cell from human body
Blood
PlasmaRBC WBC Platelets
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OBTAINING DNA FROM CELLS
Lysis of Cells
Physical / Mechanical
Grinding, Hypotonic lysis
ChemicalDetergent,
Chaotropic agents
Enzymatic
SDS,
Guanidium chloride
Proteinase K
4 -12 pH
37C
50C-60C
Tris EDTA,guanidium salts
+ SDSDisrupts structure
non covalent bonds
H2 bonds, Van der waals
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PURIFICATION OF DNA FROM CELLS
Solvent ExtractionSalting out Chromatography
CentrifugationPhenol / Chloroform
EthOH pptn
High salt conc:
Selective pptn
Gel filtration Ion Exchange Affinity Adsorption
CsCl gradient
Mol. sieve -vely charged magnetic silica / glass
Solid phase extraction
Spin column extraction
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SPIN COLUMN EXTRACTION OF DNA
Blood sample + Lysis Buffer
(Guanidine salt, Proteinase K, EthOH)
Add to silica membrane
DNA will bind to column Ptn, polysacc,
pass through
Discard
(Wash column to
remove impurities) X 2
Elute pure DNA
from column
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200l Lysis buffer
200l Blood
20l Proteinase K
65C,
10 min
200l EthOH
Mix
Load on to column
5,000g,
1 min
Discard flow through
500l Wash buffer 1
Low amount ofchaotropic salt to
remove ptns 5,000g,
1 min
Discard flow through
500l Wash buffer 2EthOH removes salts
5,000g,
1 min
Discard flow through
14,000g,
1 min
Discard flow through
Vorex
100l Elution buffer
5,000g,
1 min
Store DNA at
4C or -20C
PROCEDURE FOR DNA ISOLATION BY SPIN COLUMN
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QUANTIFICATION OF DNA
Spectrophotometric methods
Agarose gel
useful when only small quantities of nucleic acid are
available
Stained with EtBr
in aqueous solutions (TE buffer)
by measuring the absorption A (optical density, OD) in
ultraviolet light.260 nm against a blank.
proteins absorb at 280 nm.
the ratioA260/A280is used to estimate the purity of nucleic
acid.
Pure DNA should have a ratio of approximately 1.8, whereas
pure RNA should give a value of approximately 2.0.Absorption at
230 nm reflects contamination of the sample
by substances such as carbohydrates, peptides, phenols or
aromatic compounds.
In the case of pure samples, the ratioA260/A230 should be
approximately 2.2.
