Extraction of Genomic DNA

G.Umamaheswaran Ph.D Scholar JIPMER

Definition
DNA extraction is the isolation and purification of DNA

DNA in cell

Biological sources of DNA

Blood: Fresh, frozen, blood stain, blood clots
Tissues: Formalin fixed,paraffin embedded, frozen, postmortem Cells: Fixed, Cultured, buccal cells Body fluids: Amniotic, CSF, semen

DNA extraction: the basic concept

Principle
Methods
Chemical Enzymatic
SDS, sarcosyl CTAB Proteinase K Lysozyme Freezing Sonication Grinding

Cell lysis
Mechanic

Organic solvents

Phenol chloroform Sodium chloride Sodium acetate Membrane Beads Ethanol Iso-propanol

Protein removal

Salt DNA binding

DNA precipitation

Alcohol

Choice of the method depends on:

Quantity and MW of the DNA

Quality/Purity

Time
Expense

Phenol - Chloroform Method

conventional technique organic solvents

Reagents
EDTA Na2 salt RBC lysis solution Ammonium chloride Ammonium bicarbonate WBC lysis solution Na2EDTA NaCl Proteinase K SDS Saturated NaCl Buffers a) 1 X TE b) 0.5M Tris c) 0.1M Tris Equilibrated Phenol Chloroform Octanol Ethanol Hydrochloric acid Mercapto ethanol Sodium Hydroxide Pellets

Extraction Procedure
Day I

5ml of blood

Discard the plasma

Add RBC lysis solution

Mix

Centrifuge at 2500 RPM for 10

Discard the upper layer

Add RBC lysis solution, mix and centrifuge at 2500 RPM for 10 mins
Discard the upper layer

Repeat the process till RBCs are lysed completely

WBC lysis solution

Mix

SDS+ Proteinase K + MilliQ

Mix Incubate at 37o C overnight

Day II
Check for complete lysis

Add Saturated NaCl & mix

Add chloroform and mix till it becomes homogenous

Centrifuge at 4000 RPM at 4o C for 20

Separate the upper layer & transfer to a fresh tube

Add equilibrated phenol & mix

Centrifuge at 4000 RPM for 20 min at 4o C

Separate the upper layer & transfer to a fresh tube

Add Chloroform: Octanol & mix

Separate the upper layer & transfer to a fresh tube

Add ice-cold absolute ethanol & mix

DNA precipitates

Remove DNA & wash with 70% ethanol

Take out DNA from ethanol & air dry

Transfer the air dried DNA to TE buffer

Mix well & incubate the at 37o C overnight

Store the DNA at 40C

Extraction of DNA from buccal cells using kit

Easy and rapid method Time and discomfort required for sample collection Sample collection swabs - a soft foam swab on a flexible plastic handle

Collecting buccal cells

Insert the swab and rotate

Rotate swab in DNA extraction solution

Heat at 65oC for 30

DNA Sample ready for PCR

DNA extraction from paraffin embedded tissues by boiling method

Tissues formalin fixed embedded in Paraffin Wax
To deparaffinize

Xylene

Procedure
Paraffin sections and mix with Xylene in a tube Incubate at RT, After deparaffinization, centrifuge to remove xylene Wash the cell pellet with Absolute Ethanol to remove xylene

To the dry cell pellet, add cell lysis buffer, proteinase-K and SDS
Incubate overnight at 37C for complete cell lysis Heat at 97C for 10 & centrifuge for 5 Remove the supernatant (DNA) into a new tube.

DNA storage
DNA can be stored at 2 to 8C DNA precipitated in ethanol can be stored indefinitely at -20C Long term -80C Shearing of DNA - exposed to repeated freeze-thaw cycles

Precautions
Avoid exposure to infectious agents EDTA or Citrate anticoagulant is preferred Blood samples older than one week may produce poor yields

New, sterile disposable plastic tubes or autoclaved glassware should be used

All the reagents should be freshly prepared and autoclaved Polypropylene tubes and tips should be used for isolating DNA; other plastic products may absorb DNA

Downstream Applications
After DNA is extracted, it is used as a template in further molecular techniques PCR RFLP Southern Blotting RAPD AFLP RT-PCR Sequencing

DNA Analysis
Downstream techniques can:
Reveal how organisms are related Identify cryptic species Locate mutations in DNA

Thank you