Principle behind DNA isolation and roles of reagents used in isolation

1. DNA & PLASMID ISOLATION
VPB-321: Animal Biotechnology
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2. BASICS
•What is DNA?
• What is the DNA made up of?
• What are the chemical properties of DNA?
• Where is the DNA located?
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3. WHAT IS DNA?
Deoxyribonucleic Acid
 The blueprint for the life.
 It is a molecule that encodes the
genetic instructions used in the
development and functioning of all
known living organisms .
 DNA is a polymer or string of
nucleotides.
 Nucleotide is composed of a sugar,
a nitrogen base and a phosphate
backbone.
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5. WHAT IS THE DNA MADE UP OF?
Sugar MoietyVPB 321

6. BONDS & BONDS
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7. WHAT ARE THE CHEMICAL PROPERTIES OF DNA?
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8. Stacking
interactions
Charge repulsion
Chargerepulsion
The forces affecting the stability of the
DNA double helix includes:
Hydrophobic interactions - at outside
and inside helps in stabilizing structure.
Stacking interactions - these vander
Waals forces are weak yet stabilizes the
double helix.
Hydrogen bonding - Faciliates stacking.
Electrostatic interactions- DESTABILIZES
This electrostatic interaction is primarily
due to negative phosphates.
It affects intrastrand and interstand
inetractions. But can be neutralized with
positive charges e.g., Na+ ions or
CHEMISTRY OF DNA
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9. Where is/are the DNA located?
Nucleus
Mitochondria
Plastids
Bacterial nucleoid
Plasmids
Plasmids
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10. .
Genomic DNA
(SINGLE COPY)
Plasmid DNA
(MULTIPLE COPY)
1. Genomic DNA is chromosomal DNA where the
genetic material is present.
1. Plasmid DNA is extra-chromosomal DNA in bacteria
and some yeasts, i.e. Plasmid.
2. It is primary DNA in all living organisms. 2. It is secondary DNA.
3. It is linear in eukaryotes whereas circular in
prokaryotes
3. It is circular.
4. As it encodes genetic information, it is much larger
than plasmid.
4. It is smaller.
5. Genomic DNA is organized with proteins called
histones.
5. Plasmid DNA is not with histones.
6. It contains essential genes which codes for
functional and structural proteins.
6. It contains non-essential genes.
7. It can be transferred only through cell division
within the same species.
7. It can be transferred through horizontal way of gene
transfer between same or different species.
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11. HOW TO TAKE THIS DNA OUT OF THE CELL?
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12. DNA ISOLATION vs DNA EXTRACTION
DNA ISOLATION
Isolation aims to get as much of the
target out of your sample as possible.
Along the step of isolation one usually
picks up quite a bit of contamination.
DNA EXTRACTION
Extraction is just one specific way to
achieve isolation and purification.
Extraction uses a solvent that serves as
extractant and can be devided in above
stages:
1) Gentle lysis of the cells / solubilization of
DNA or isolation
2) Removal of contaminants (proteins, RNA
and other macromolecules) or the so-
called purification is achieved either by
enzymatic or chemical means.
https://www.researchgate.net/
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13. DIFFERENT SOURCES FOR DNA ISOLATION
 DNA can be isolated from all the types living cells and fossilsized cells
containing nucleus or nucleiod or semi autonomous organelles. The
viral DNA can also be isolated.
 For example: Blood, other body fluids, bacterial culture etc.
 We will overview: Genomic DNA isolation from blood, plasmid DNA
isolation and bacterial genomic DNA isolation
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14. Genomic DNA Extraction
SOURCE: MICROBES & BLOOD
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15. BASIC DIFFERENCES
 Genomic DNA (gDNA) extraction is the simpler procedure because strong lysis is
the only step necessary to release gDNA into solution. For yeast, plants and
bacteria, lysis involves enzymatically breaking the strong, rigid cell wall before
mechanically disrupting the plasma membrane.
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16. STEPS
 STEPS:
1. Lysis or cells disruption
2. Phase seperation
3. Clearing proteins
4. Precipitating DNA
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17. 1. Lysis or cells disruption
Extraction buffer and lysis buffer and incubation at 65°C.
These extraction/ lysis buffer contains a high concentration of chaotropic salts.
Firstly, they destabilize hydrogen bonds, van der Waals forces and hydrophobic interactions, leading to
destabilization of proteins, including nucleases. Secondly, they disrupt the association of nucleic acids
with water.
NaCl : phosphate of DNA molecule repel one molecule from others. Na+ ions form an ionic bond
with phosphates and neutralized the negative charge allowing DNA molecules grouping.
EDTA (Ethylenediamine tetraceticacid): chelating agent with high affinity to metallic ions of Mg,
DNAse cofactors (enzymes that degrade the DNA). EDTA bind to ions and overturn its effects.
CTAB (Hexadecyl trimethyl-ammonium bromide): detergent used to break cellular membranes
and remove lipids
Other stabilizer agents : Protinase-K, Tris HCl, sorbitol, sodium bisulphite, DTT, detergents: SDS
(remove lipids), sarkosyl, triton, PVP (bind to polyphenols- components of vegetable cellular
wall-removing), 2-mercaptoethanol (denature proteins).
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18. GRAM NEGATIVE VS GRAM POSITIVE - LYSIS
 The presence of a high peptidoglycan content in
the cell walls of Gram positive bacteria is a major
hurdle in the isolation of DNA.
 So it’s resistance to conventional methods of lysis .
 Lysozyme + detergents such as SDS ensures better
lysis of the Gram positive cell wall .
 Penicillins* which is known for interfering in the
assembly of N-Acetylglucosamine (NAG) and N-
Acetylmuramic acid (NAM) moieties during Gram
positive cell wall synthesis can also solve the
purpose.
*De, Sachinandan et al. “A Simple Method for the Efficient Isolation of Genomic DNA from Lactobacilli Isolated from Traditional Indian Fermented
Milk (Dahi).” Indian Journal of Microbiology 50.4 (2010): 412–418. PubMed Central. Web.
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19. BLOOD DNA ISOLATION -LYSIS
 Enzymes may also feature LYSIS
of RBC and WBC in presence of
lysisbuffer.
 The broad-spectrum serine
protease proteinase K is also
very efficient in digesting
proteins away from nucleic acid
preparations.
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20. 2. PHASE SEPARATION
Addition of chloroform-isoamylalcohol
(24:1) or phenol- chloroform
isoamylalcohol (25:24:1). Centrifugation.
Organic solvents, hydrophobics lysates
keep trapped, eg. membrane lipids,
proteins or polysacharids. Besides
denature proteins.
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21. ROLE OF PHENOL
 DNA is negatively charged molecule, therefore soluble in
polar solvent like water. Whereas proteins has both polar
(hydrophilic) and non polar (hydrophobic) side chains amino
acids.
 In cell normally the DNA remain dissolved in polar phase and
the proteins tend to fold in such a way that their hydrophilic
or polar groups face outward in solvent and the hydrophobic
toward inside.
 On adding phenol, proteins flip since phenol is less polar
than water.
 Now, therefore protein (also lipids) becomes soluble in
phenol or non polar phase and DNA in water phase. In such a
way we acheive separation of DNA from proteins (lipids).
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22. ROLE OF PHENOL & ISOAMYL ALCOHOL
 Chloroform is significantly
denser than water, so adding it
to the organic phase increases
the overall density of that
phase, helping to prevent
phase inversion.
 Sometimes, the choloroform
when added next to the P:C
treatment, helps in extracting
phenol from the aqueous
solution
 Isoamyl alcohol acts as anti-
foaming agent.
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23. 3. CLEARING OUT
PROTEINS
 Proteins are eliminated adding a
protease and increasing the
osmolarity (sodium acetate or
ammonium acetate)
 DNA precipitates with alcohol –
usually pure and could ethanol or
isopropanol (2-propanol). Because
DNA is non-soluble in alcohol,
precipitate and form a pellet in the
bottom of the tube after
centrifugation.
 This step also remove alcohol soluble
salts.
 DNA cleans with 70% ethanol, dry
and dilute in TE buffer (protect DNA
from degradation) or sterile distilled
water.
4. PRECIPITATING
DNA
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24. BLOOD DNA ISOLATION PROTOCOL
GENOMIC DNA ISOLATION FROM HUMAN WHOLE BLOOD SAMPLES BY NON ENZYMATIC
SALTING OUT METHOD. SUGUNA et. al. International Journal of Pharmacy and Pharmaceutical Sciences. Vol 6, Issue 6, 2014VPB 321

25. cont..
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26. MICROBIAL DNA ISOLATION PROTOCOL
He, Fanglian. “E. Coli Genomic DNA Extraction.” BIO-PROTOCOL 1.14 (2011): n. pag. CrossRef. Web. 8 Apr. 2017.VPB 321

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29. PLASMID DNA
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30. STEPS
 Plasmid DNA extraction is a bit
trickier because plasmid DNA must
be kept separate from gDNA.
 This separation is based on size, and
good separation relies on using the
right lysis method.
 STEPS:
1. ALKALINE Lysis or cells disruption
2. Phase seperation
3. Clearing proteins
4. Precipitating DNA
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31. ALKALINE LYSIS?
 For plasmid DNA extraction, the lysis has to be a lot more subtle. Firstly
perform a SDS bases lysis as done in case of gDNA.
 Next the sample is neutralized in a potassium acetate solution to
renature the plasmid, and this is key to the separation of plasmid and
gDNA.
 Because plasmids are small, they can easily reanneal forming dsDNA.
 Genomic DNA, however, is too long to reanneal fully, and instead it tends
to tangle so that complimentary strands remain separated.
 During centrifugation, gDNA (bound to protein) forms a pellet while
plasmid DNA remains soluble.
 It is key at this step not to vortex or mix the sample vigorously because
gDNA breaks easily, and broken gDNA may be small enough to reanneal
and go into solution with the plasmid.VPB 321

32. PLAMSID DNA ISOLATION PROTOCOL
He, Fanglian. “Plasmid DNA Extraction from E. Coli Using Alkaline Lysis Method.” BIO-PROTOCOL 1.1 (2011): n. pag. CrossRef. Web. 8 Apr.
2017.
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34. DNA QUANTIFICATION
HOW TO CHECK DNA THAT YOU EXTRACTED?
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35. 1. Electrophoresis in agarose gels- qualitative
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36. 2. Espectrophotometer/ nanometer- quantitative
 Gives the 260/ 280 ratio which
tells us about purity of DNA.
260/280 = 1.8 stable
260/280 = 2.0 >
(contaminated with proteins)
260/280 = 1.6 <
(contaminated with RNA)
 Also it quantifies and displays
concentration of DNA in ng/ul.
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37. THANK YOU
QUERIES?
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