GENOMIC DNA EXTRACTION & STBP2033

DETERMINATION
What is genomic DNA (gDNA)?

Purpose of gDNA extraction

WHAT WILL WE
Methods in gDNA extraction BE LEARNING
TODAY?
Evaluation of gDNA

Troubleshooting tips
WHAT IS GENOMIC DNA?
The genome of an organism (encoded by the genomic DNA) is the
(biological) information of heredity which is passed from one
generation of organism to the next. Researchers refer to DNA
found in the cell's nucleus as nuclear DNA. An organism's complete
set of nuclear DNA is called its genome.
Did you know?
gDNA is chromosomal DNA,
in contrast to extra-
chromosomal DNAs like
plasmids.
WHERE CAN GENOMIC DNA BE FOUND?
WHY DO WE EXTRACT GENOMIC DNA?
To obtain DNA in a relatively purified form which can be used for
further investigations such as Polymerase chain reaction (PCR),
Restriction fragment length polymorphism (RFLP), Southern Blotting
that are used for:
Screening of genetic diseases
Determination of paternity
Analysis of forensic evidence
Development of diagnostics and drugs
METHODS FOR GENOMIC DNA EXTRACTION
Most DNA extraction protocols consist of two parts:
A technique to lyse the cells gently and solubilize the DNA
Enzymatic or chemical methods to remove contaminating proteins,
RNA, or macromolecules
Most commonly used DNA extraction procedures:
Organic (Phenol-chloroform) extraction
Non-organic (Proteinase K and salting out)
Adsorption method (silica-gel membrane)
ORGANIC EXTRACTION
The most basic of all procedures
in molecular biology
Typical involves these steps :
i. Cell lysis
ii. DNA purification
iii. Washing and precipitation with
alcohol
1. CELL LYSIS
In order to extract DNA from a tissue/cells of interest, the cells must be separated
and the cell membranes have to be disrupted.
This process is carry out by lysis buffer, also known as detergent – designed to lyse
outer cell membrane and nuclear membrane
Some examples of detergents :
 EDTA (Ethylenediaminetetraacetic disodium salt) – chelating agent of divalent cations such as Mg2+ -
essential for preserving the overall structure of the cell membrane
 SDS (sodium dodecyl sulfate) – aids in disrupting the cell membranes by removing its lipid
 CTAB (cetyl trimethylammonium bromide) – facilitates the separation of polysaccharides – widely used
in DNA extraction from plant tissues

Cell debris and partially digested organelles – pelleted by centrifugation leaving the
cell extract as a reasonably clear supernatant.
HOW DOES DETERGENT WORKS?
Breaks apart membranes by attaching to the lipids & proteins in the membranes.

Once the cell and nuclear membranes have been broken apart, as well as all of the
organelle membranes such as mitochondria and chloroplasts, so what is left?
 Proteins
 Carbohydrates
 DNA
2. DNA PURIFICATION
In addition to DNA, the cell extract will contain significant quantities of detergents,
salts and reagents used during cell lysis step, proteins and RNA.
These can be removed by :
 Phenol-chloroform
 Standard way to remove proteins from nucleic acids solution
 Phenol denatures proteins in the sample. After centrifugation, denatured
proteins remain in the interphase.
 Aqueous phase containing nucleic acid is mixed with chloroform that removes
phenol residues from the solution.
 Proteinase K
 Proteolytic enzyme to digest most proteins present in the solution
 RNAse
 To degrade RNA that might present in the solution
3. DNA PRECIPITATION
Most widely used method in precipitation of DNA is with ice-cold
ethanol/isopropanol.
 Precipitation of DNA is improved by increasing ionic strength, usually by
adding sodium acetate.
 The salt interrupt the H bonds between water and DNA molecules.
 In the presence of cations, ethanol induces a structural change in DNA
molecules that causes them to aggregate and precipitate out of solution,
giving a pellet upon centrifugation.
Solutes that may be trapped in the precipitate may be removed by washing
the DNA pellet with a solution of 70% ethanol.
 Before After

Supernatant 70% EtOH

Centrifuge Wash Centrifuge

Pellet

Dissolve pellet
(H2O, TE, etc.)
• Pellet down nucleic acids.
Add alcohol and salt to
• Wash pellet with 70% ethanol to remove
precipitate nucleic acids from the
residual salts and other contaminants.
aqueous fraction
• Discard ethanol and allow pellet to dry.
 Non-organic extraction
 Does not use organic reagents such as phenol or chloroform
 Digested proteins are removed by salting out with high
concentration of LiCl or NaCl
Adsorption method
 Crude lysate is prepared by mixing sample with extraction buffer
OTHER DNA – low pH and high ionic strength
 This is then applied to a column with silica-gel membrane
EXTRACTION  Nucleic acid will bind to the membrane, while contaminants pass

METHODS through the column

 Residual contaminants are washed away using wash buffer and
nucleic acid on the membrane will be eluted using elution buffer –
high pH and low ionic strength
EVALUATION OF GENOMIC DNA
Why do we need to evaluate the genomic DNA that you have
extracted?
Determine the quality and quantity of gDNA
Poor quality DNA will affect your subsequent experiments
The quality and quantity of extracted gDNA can be measured
using:
UV absorbance spectrometry
Agarose gel electrophoresis
UV ABSORBANCE SPECTROMETRY
The amount of UV radiation absorbed by a solution of DNA is directly proportional to the
amount of DNA sample.
Nucleic acids have an absorption peak at 260 nm.
For quantification of DNA :
 [dsDNA] ≈ A260 x (50 µg/mL)
 [ssDNA] ≈ A260 x (33 µg/mL)

For DNA quality :

 A260/A280 ratio
 Optimum : 1.8 – 2.0
 Lower than 1.7 indicating protein contamination
 A260/A230 ratio
 Optimum : 1.8 – 2.0
 Lower than 1.7 indicating organic compound contamination (e.g. phenol, alcohol, or carbohydrates)
AGAROSE GEL ELECTROPHORESIS
What is gel electrophoresis of DNA?
 Separation of DNA fragments according to size, based on movement through a gel medium when an
electric field is applied

Agarose
 A polysaccharide made from seaweed
 Dissolved in buffer and heated, then cools to a gelatinous solid with a network of crosslinked molecules

Agarose gel electrophoresis is use commonly to examine the integrity of the DNA
 Good quality DNA should migrate as a high molecular weight band, with little or no evidence of
smearing.
 Smearing indicates degradation of DNA
HOW SHOULD A GOOD DNA EXTRACTION LOOKS
LIKE?
Single high molecular
weight band
Severe
degradation
High molecular weight of DNA, best
band with smearing. to re-extract
These samples are
fine and can be used
for PCR

No smearing

PERFECT!! NOT BAD LAR ☺

 TROUBLESHOOTING TIPS
Problem Cause
Low DNA yield Incomplete lysis
Low DNA yield / DNA is Bubbles formed during
sheared or degraded mixing steps
Lysate mixed too vigorously
or small pipette tips used
during mixing
DNA is sheared or DNA repeatedly frozen and
degraded thawed
DNA contaminated with
DNAses
Solution
•Decrease the amount of starting material used.
•Be sure to add Proteinase K during lysis.
•Increase the length of incubation at room
temperature.
Make sure that the pipette tip is submerged in the
solution during mixing
•Use the appropriate pipette tip set to a volume
lower than the total volume of solution in the
sample.
•Pipet up and down gently to mix.
Aliquot DNA and store at 4°C or -20°C. Avoid
repeated freezing and thawing.
Maintain a sterile environment while working
(i.e. wear gloves and use DNase-free reagents).