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
