DNA and RNA extraction

1. DNA and RNA Extraction
Prof. GHIZAL FATIMA
ERA UNIVERSITY,
LUCKNOW

2. • DNA isolation is a process
of purification of DNA from sample using a
combination of physical and chemical methods. The
first isolation of DNA was done in 1869 by Friedrich
Miescher. Currently it is a routine procedure in
molecular biology or forensic analyses.
• For the chemical method, there are many different
kits used for extraction, and selecting the correct one
will save time on kit optimization and extraction
procedures. PCR sensitivity detection is considered
to show the variation between the commercial kits

3. BASIC PROCEDURE
• There are three basic and two optional steps in a DNA
extraction:
• Cells which are to be studied need to be collected.
• Breaking the cell membranes open to expose the DNA
along with the cytoplasm within (cell lysis).
– Lipids from the cell membrane and the nucleus are broken
down with detergents and surfactants.
– Breaking proteins by adding a protease (optional).
– Breaking RNA by adding an RNase (optional).
• The solution is treated with concentrated salt solution
to make debris such as broken proteins, lipids and RNA
to clump together.
• Centrifugation of the solution, which separates the
clumped cellular debris from the DNA.

4. • DNA purification from detergents, proteins, salts and reagents
used during cell lysis step. The most commonly used
procedures are:
– Ethanol precipitation usually by ice-cold ethanol or isopropanol.
Since DNA is insoluble in these alcohols, it will aggregate together,
giving a pellet upon centrifugation. Precipitation of DNA is
improved by increasing of ionic strength, usually by adding sodium
acetate.
– Phenol–chloroform extraction in wich phenol denatures proteins
in the sample. After centrifugation of the sample, denaturated
proteins stay in the organic phase while aqueous phase
containing nucleic acid is mixed with the chloroform that removes
phenol residues from solution.
– Minicolumn purification that relies on the fact that the nucleic
acids may bind (adsorption) to the solid phase (silica or other)
depending on the pH and the salt concentration of the buffer.

5. • Cellular and histone proteins bound to the DNA
can be removed either by adding a protease or by
having precipitated the proteins
with sodium or ammonium acetate, or extracted
them with a phenol-chloroform mixture prior to
the DNA-precipitation.
• After isolation, the DNA is dissolved in slightly
alkaline buffer, usually in the TE buffer (The TE
Buffer is used for protection of DNA and RNA from
degradation. The Tris buffering agent and EDTA metal
chelating properties help protect DNA and RNA), or
in ultra-pure water.

6. Special types
• Specific techniques must be chosen for
isolation of DNA from some samples. Typical
samples with complicated DNA isolation are:
• archaeological samples containing partially
degraded DNA.
• samples containing inhibitors of subsequent analysis
procedures, most notably inhibitors of PCR, such
as humic acid from soil, indigo and other fabric dyes
or haemoglobin in blood
• samples from microorganisms with thick
cellular wall, for example yeast

7. • Extrachromosomal DNA (DNA that is found outside the
nucleus of a cell. It is also referred to as extranuclearDNA or
cytoplasmic DNA. Most DNA in an individual genome is found
in chromosomes but DNA found outside the nucleus also
serves important biological functions) is generally easy to
isolate, especially plasmids may be easily isolated by cell lysis
followed by precipitation of proteins, which traps
chromosomal DNA in insoluble fraction and after
centrifugation, plasmid DNA can be purified from soluble
fraction.
• A Hirt DNA Extraction is an isolation of all extrachromosomal
DNA in a mammalian cell. The Hirt extraction process gets rid
of the high molecular weight nuclear DNA, leaving only low
molecular weight mitochondrial DNA and any
viral episomes present in the cell.

8. Detecting DNA
• A diphenylamine (DPA) indicator confirms the presence
of DNA. This procedure involves chemical hydrolysis of
DNA: when heated (e.g. ≥95 °C) in acid, the reaction
requires a deoxyribose sugar and therefore is specific
for DNA.
• Under these conditions, the 2-deoxyribose is converted
to w-hydroxylevulinyl aldehyde, which reacts with the
compound, diphenylamine, to produce a blue-colored
compound. DNA concentration can be determined
measuring the intensity of absorbance of the solution
at the 600 nm with a spectrophotometer and
comparing to a standard curve of known DNA
concentrations.

9. • Measuring the intensity of absorbance of the
DNA solution at wavelengths 260 nm and 280
nm is used as a measure of DNA purity.
• DNA absorbs UV light at 260 and 280 nms, and
aromatic proteins absorb UV light at 280 nm; a
pure sample of DNA has a ratio of 1.8 at 260/280
and is relatively free from protein contamination.
• A DNA preparation that is contaminated with
protein will have a 260/280 ratio lower than 1.8.

10. • DNA can be quantified by cutting the DNA with
a restriction enzyme, running it on an agarose gel,
staining with ethidium bromide or a different stain and
comparing the intensity of the DNA with a DNA marker
of known concentration.
• Using the Southern blot technique, this quantified DNA
can be isolated and examined further
using PCR and (Restriction Fragment Length
Polymorphism) RFLP analysis.
• These procedures allow differentiation of the repeated
sequences within the genome. It is these techniques
which forensic scientists and molecular biologist use
for comparison, identification, and analysis.

12. Q1-What are the three main steps of DNA extraction?
A1-Step 1: Lysis. In this step, the cell and the nucleus are broken
open to release the DNA inside and there are two ways to do
this. ...
• Step 2: Precipitation. Ethanol precipitation is a commonly used
technique for concentrating and de-salting nucleic acids (DNA or RNA)
preparations in aqueous solution. The basic procedure is that salt and
ethanol are added to the aqueous solution, which forces the precipitation of
nucleic acid out of solution.
• Step 3: Purification.
• Q2-Why is DNA extracted from human cells?
• DNA is extracted from human cells for a variety of
reasons. With a pure sample of DNA you can test a
newborn for a genetic disease, analyze forensic evidence,
or study a gene involved in cancer etc.

13. Q3-What is the function of sodium dodecyl sulphate in a DNA extraction?
A3-Sodium Dodecyl Sulfate, is a detergent that is known to denature proteins. ... It is
also used in nucleic acid extraction procedures for the disruption of cell walls and
dissociation of nucleic acid:protein complexes.
Q4-Can DNA be extracted from urine?
Urine is not considered an ideal source of DNA due to the low concentration of
nucleated cells present in human urine. The nucleated cells found in urine are
typically white blood cells and epithelial cells.
Q5-What is the importance of DNA extraction?
A-5- Studying the genetic causes of disease and for the development of diagnostics
and drugs. It is also essential for carrying out forensic science, sequencing
genomes, detecting bacteria and viruses in the environment and for determining
paternity.
Q6-Why do you use alcohol in DNA extraction?
A6-Since DNA is insoluble in ethanol and isopropanol, the addition of alcohol,
followed by centrifugation, cause the DNA proteins to come out of the solution.
When DNA concentration in the sample is heavy, the addition of ethanol will cause
a white precipitate to form immediately.

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. Several methods are used in molecular biology to
isolate RNA from samples, the most common of these
is Guanidinium thiocyanate-phenol-chloroform
extraction.
• The filter paper based lysis and elution method
features high throughput capacity.
• RNA extraction in liquid nitrogen, commonly using a
mortar and pestle (or specialized steel devices known
as tissue pulverizers) is also useful in preventing
ribonuclease activity.

15. • RNA (Ribonucleic acid) is a polymeric substance present in
living cells and many viruses, consisting of a long single-
stranded chain of phosphate and ribose units with
the nitrogen bases adenine, guanine, cytosine, and uracil,
which are bonded to the ribose sugar.
• RNA is used in all the steps of protein synthesis in all living
cells and carries the genetic information for many viruses.
• The isolation of RNA with high quality is a crucial step
required to perform various molecular biology experiment.
• TRIzol Reagent is a ready-to-use reagent used for RNA
isolation from cells and tissues.

16. • TRIzol works by maintaining RNA integrity
during tissue homogenization, while at the
same time disrupting and breaking down cells
and cell components.
• Addition of chloroform, after the
centrifugation, separates the
solution into aqueous and organic phases.
RNA remains only in the aqueous phase.

17. • After transferring the aqueous phase, RNA can be
recovered by precipitation with isopropyl alcohol.
• But the DNA and proteins can recover by sequential
separation after the removal of aqueous phase.
• Precipitation with ethanol requires DNA from the
interphase, and an additional precipitation with isopropyl
alcohol requires proteins from the organic phase.
• Total RNA extracted by TRIzol Reagent is free from the
contamination of protein and DNA.
• This RNA can be used in Northern blot analysis, in vitro
translation, poly (A) selection, RNase protection assay, and
molecular cloning.

18. Procedure
• Materials Required
• Reagents
• Chloroform (without any additives, such as isoamyl alcohol)
• Isopropyl alcohol
• 75% Ethanol (in DEPC-treated water)
• RNase-free water or 0.5% SDS solution
• The SDS solution must be prepared using DEPC-treated,
autoclaved water. DEPC inactivates the RNases by the
covalent modifications of the histidine residues.

19. • Homogenization
• Growth medium on the cells was discarded
and cells were washed with ice cold 1X PBS.
The monolayer was then covered with 1 ml of
l TRIzol and the cells were lysed and
homogenized by repeated pipetting.

20. • Phase Separation
•
• The homogenized samples were incubated for 5 minutes at 15 to
30°C for the complete dissociation of nucleoprotein complexes.
• 0.2 ml (200 microliters)of chloroform per 0.75 ml of TRIZOL LS
Reagent was added. The tubes were shaked vigorously by hand for
15 seconds and incubated them at 15 to 30°C for 2 minutes.
• The samples were centrifuged for 15 minutes at no more than
12,000 g (4°C).
• The aqueous phase was transferred to other tubes. ( Following
centrifugation, the mixture separates into a lower red, phenol-
chloroform phase, an interphase, and a colorless upper aqueous
phase. RNA remains only in the aqueous phase. The volume of the
aqueous phase is about 70% of the volume of TRIZOL LS Reagent
used for homogenization.)

21. • RNA Precipitation
• The RNA was precipitated from the aqueous
phase by mixing with 3 microlitre of glycogen
and 500 microlitre of isopropyl alcohol.
• The mixture was centrifuged for 30 minutes at
12,000 × g (2 to 8°C).( The RNA precipitate
forms a gel-like pellet on the side of the tube
at bottom).

22. • RNA Wash
• The supernatant was removed. The RNA pellet
was washed once with 75% ethanol, adding 900
microlitre of 75% ethanol per 0.75 ml of TRIZOL
LS Reagent used for the initial homogenization.
• The sample were inverted and mixed and
centrifuged at 12,000 rpm for 30 minutes at 4
degree.

23. • Redissolving RNA
• The RNA pellet was dried .
• RNA was dissolved in RNase-free water (or 0.5% SDS
solution) by passing the solution through the pipette
tip for a few times, and incubating for 10 minutes at 55
to 60°C.
• Note: 0.5% SDS should not be used if RNA will undergo
further enzymatic reaction. DNA can also be in 100%
formamide (deionized) and stored at -70°C.

24. Precautions for preventing RNase contamination:
• RNases can be introduced into the RNA preparation at any point
accidently during the isolation procedure through improper
technique.
• RNase activity is difficult to inhibit, so it is very essential to prevent
its introduction. The following guidelines should be observed
carefully while working with RNA.
• Always wear disposable gloves. Usually our skin contains many
bacteria and molds that can contaminate the RNA preparation and
be a source of RNases.
• Good and clean microbiological techniques will prevent microbial
contamination. Use sterile, disposable plastic ware and automatic
pipettes reserved for RNA work to prevent cross-contamination
with RNases from shared equipment.

25. Q1-Why trizol is used for RNA isolation?
• TRIzol Reagent is a ready-to-use reagent used for RNA
isolation from cells and tissues.TRIzol works by
maintaining RNA integrity during tissue homogenization,
while at the same time disrupting and breaking down cells
and cell components. ...RNA remains only in the aqueous
phase.
Q2-What is RNA extraction used for?
• This procedure is complicated by the ubiquitous presence of
ribonuclease enzymes in cells and tissues, which can rapidly
degrade RNA.
Q3-What does chloroform do in RNA isolation?
• After solubilization, the addition of chloroform causes phase
separation (much like extraction with
phenol:chloroform:isoamyl alcohol), where protein
is extracted to the organic phase, DNA resolves at the
interface, and RNA remains in the aqueous phase.