5. What is DNA Extraction?
A routine procedure to collect DNA for subsequent molecular or
forensic analysis.
DNA Extraction is the isolation and purification of DNA
(deoxyribonucleic acid)
6. What is it used for?
Extraction of DNA is often an early step in
many diagnostic processes used to detect
bacteria and viruses in the environment as
well as diagnosing disease and genetic
disorders.
7. Lysis of the cells
Removal of contaminants
Proteins
RNA
Other macromolecules
Concentration of purified DNA (if required)
8. Steps to DNA Extraction
1. Breaking the cells open to expose DNA
2. Remove membrane lipids by adding
detergent
3. Precipite DNA with an alcohol usually
ethanol or isopropanol. Since DNA is
insoluble in these alcohols, it will
aggregate together, giving a pellet upon
centrifugation. This step also removes
alcohol-soluble salt.
9. How does it work?
1- Break open (lyse) the cells or virus
containing the DNA of interest This is often
done by sonicating or bead beating the
sample. Vortexing with phenol (sometimes
heated) is often effective for breaking down
protienacious cellular walls or viral capsids.
The addition of a detergent such as SDS is
often necessary to remove lipid membranes.
10. How does it work?
2- DNA associated proteins, as well as other
cellular proteins, may be degraded with the
addition of a protease. Precipitation of the
protein is aided by the addition of a salt such as
ammonium or sodium acetate. When the sample
is vortexed with phenol-chloroform and
centrifuged the proteins will remain in the
organic phase and can be drawn off carefully.
The DNA will be found at the interface between
the two phases.
11. How does it work?
3- DNA is the precipitated by mixing with cold ethanol
or isopropanol and then centrifuging. The DNA is
insoluble in the alcohol and will come out of solution,
and the alcohol serves as a wash to remove the salt
previously added.
4- Wash the resultant DNA pellet with cold alcohol
again and centrifuge for retrieval of the pellet.
5- After pouring the alcohol off the pellet and drying, the
DNA can be re-suspended in a buffer such as Tris or
TE.
6- Presence of DNA can be confirmed by
electrophoresing on an agarose gel
containing ethidium bromide, or another
fluorescent dye that reacts with the DNA, and
checking under UV light.
12. RNA
RNA molecule contains the 5-carbone sugar
ribose in its structure, instead of deoxyribose
which exists in DNA molecule.
RNA is more unstable than DNA (more
sensitive to action of hydrolases)
Ribonucleases (Rnases) are very stable and
active enzymes without cofactors
14. RNA extraction
RNA extraction is the purification of RNA
from biological samples. This procedure is
complicated by the ubiquitous presence of
ribonuclease enzymes in cells and tissues,
which can rapidly degrade RNA.This method
often uses a proprietary formulation of this
reagent called Trizol.
15. RNA Extraction Methods
Trizol solution: must
be used under fume
hood
extraction method:
difficult and not
practical
Qiagen extraction kits:
user-friendly and
efficient
Best ways of extraction
16. Spectrophotometer Calculation of
RNA & DNA
Quartz Cuvettes
RNA & DNA extracted sample:
OD in 260nm × 40 × dilution = RNA μg/ml
OD in 260nm × 50 × dilution = DNA μg/ml
abs260
Purity = abs
280
For DNA 1.8 – 2.0 very good
less than 1.8 presence of protein contamination
