Practical No : 13

Practical : LIVAK DNA Extraction Protocol

Materials :
1.6 mL 5M NaCl
5.48g sucrose
1.57g Tris
10.16 mL 0.5M EDTA
2.5 mL (20% SDS)
14 µL 8M K-acetate
200 µL 99% ethanol
70% ethanol

Procedure:
LIVAK Grind buffer preparation

1.6 mL 5M NaCl, 5.48g sucrose, 1.57g Tris, 10.16 mL 0.5M EDTA and 2.5
mL (20% SDS) were mixed with each other and the total volume was brought up to
100ml. Then the solution was filter sterilized and was stored at -20ºC. Next the solution
was heated in water bath and finally the solution was mixed before each use to re-
dissolve the precipitate.

DNA extraction from mosquito

A mosquito was ground in 100 µL preheated grind buffer in 1.5 mL Eppendorf

and was transferred immediately to 65ºC. Then the solution was incubated at 65ºC for
approximately 30 minutes. Next the solution was microcentrifuged briefly to collect
condensation. Then 14 µL 8M K-acetate was mixed to obtain final concentration 1M
and was incubated on ice approximately 30 minutes. After that the solution was
centrifuged 10 minutes at 13,000 rpm and was transferred the supernatant to new 1.5
mL Eppendorf without transferring the debris. Then 200 µL 99% ethanol as added, was
spun and was incubated 2 minutes at room temperature. Next the solution was
centrifuged 13,000 rpm for 5 minutes and the supernatant was removed. After that the
pallet was rinsed in approximately 100 µL ice cold 70% ethanol for 2 times being
careful not to dislodged pellet and then the supernatant was removed. Next the pellet
 was dried and open tubes were left on bench top approximately for 1 hour. Finally the
pellet was suspended in 100 mL distilled water and was incubated at 65ºC for 10
minutes.

Observation:

Eppendorf

Extracted DNA from a mosquito

Discussions:
It is quite clear that the extraction methods have to be adapted in such a way that they can
efficiently purify DNA from various sources. Another important factor is the sample size. If
the sample is small (for example sperm, or a single hair) the method has to be different to the
method used in isolating DNA from a couple of milligrams of tissue or milliliters of blood.
Another important factor is whether the sample is fresh or has been stored. Stored samples can
come from archived tissue samples, frozen blood or tissue, exhumed bones or tissues, and
ancient human, animal, or plant samples.

The isolation of DNA usually begins with lysis, or breakdown, of tissue or cells. This process
is essential for the destruction of protein structures and allows for release of nucleic acids from
the nucleus. Lysis is carried out in a salt solution, containing detergents to denature proteins or
proteases (enzymes digesting proteins), such as Proteinase K, or in some cases both. It results
in the breakdown of cells and dissolving of membranes.

While the lysis of soft tissues or cells is easy, DNA also has to be isolated from hard tissues,
such as bone, wood, and various plant materials. Most plant samples require freezing in liquid
nitrogen and subsequently pulverizing the tissues to a fine powder. On the other hand, bones
are highly mineralized and the ions have to be removed from the samples before extraction so
they do not later interfere with PCR. Once the samples are partly processed they are then
homogenized in lysis buffer using a mechanical homogenizer.

DNA isolation is a simple process and can be performed in a kitchen using household
appliances and chemicals. Vegetables or meat can be homogenized with salt and water. After
that, by application of a detergent, cellular proteins and lipids are separated away from DNA.
Enzymes found in meat tenderizer or pineapple juice allow precipitation of proteins and free
DNA into the solution. By adding alcohol to the mix, nucleic acid is brought to the top of the
container and can be spooled onto a stick as a visible white string.

DNA extraction methods follow some common procedures aimed to achieve effective
disruption of cells, denaturation of nucleoprotein complexes, inactivation of nucleases and
other enzymes, removal of biological and chemical contaminants, and finally DNA
precipitation. Most of them follow similar basic steps and include the use of organic and
nonorganic reagents and centrifugation methods. Finally, they have developed into a variety of
automated procedures and commercially available kits. Lysis of cells is a common step in most
DNA extraction protocols, and it is commonly achieved through the use of detergents and
enzymes. Sodium dodecyl sulfate (SDS) and Triton™ X-100 (Sigma-Aldrich, St Louis, MO,
USA) are examples of popular detergents used to solubilize cell membranes. Enzymes are also
combined with detergents to target cell surface or cytosolic components. Proteinase K is a
commonly used enzyme used in various protocols to cleave glycoproteins and inactivate
RNases and DNases. Other denaturants such as urea, guanidinium salts, and chemical
chaotropes have also been used to disrupt cells and inactivate cellular enzymes, but these can
impact on quality and nucleic acid yield.

A number of commercial DNA purification kits use the very same principles as this household
method, but different reagents. In a commercial kit the common lysis solutions contain: sodium
chloride, tromethamine (also known as Tris), which is a buffer to retain constant pH;
ethylenediaminetetraacetic acid (EDTA), which binds metal ions; and sodium dodecyl sulfate
(SDS), which is a detergent. A common enzyme used in DNA extraction is Proteinase K.

The oldest methods of DNA purification in laboratories, still often used also by the FBI , rely
on a mix of organic solvents. Lysed samples are mixed with phenol, chloroform, and
isoamylalcohol for separation of DNA and protein. Proteins are denatured by the organic
mixture. When the sample is centrifuged, DNA is retained in the aqueous (water) layer, phenol
is at the bottom of the tube, and denatured proteins form a cloudy interface. This method is
very efficient, but unfortunately it can only be used if the quantity of starting material is
reasonably abundant. Moreover, the organic solvents used carry health and safety problems.
The quality of the DNA from this procedure is usually not adequate for some more sensitive
analytical techniques (especially sequencing and occasionally PCR).

A modification of the method uses high salt (sodium chloride, NaCl) concentration to bring
down DNA. After the denaturation of cellular proteins using detergents and a protease for a
few hours or overnight, salt is added and mixed with the solution. As a result, salt of nucleic
acid is formed and in presence of alcohol can be recovered by centrifugation.

Occasionally, alkaline denaturation of the sample is used to release DNA from the cells. Buccal
swabs and occasionally blood stains can be placed in small plastic tubes (eppendorfs) and
subjected to denaturation with sodium hydroxide (NaOH). The solution is then re-equilibrated
to neutral pH with a more acidic buffer solution and is ready for PCR. Although it is a quick
and simple method, the quality of DNA is not always adequate for all applications.

A method similar to alkaline denaturation is heat denaturation, achieved by boiling samples.

Heating of a sample to 100°C releases DNA into the solution but also denatures it by separating
the two strands. In some cases this procedure gives adequate nucleic acid that can be amplified
by PCR, however, most of the time there are remaining inhibitors in the form of degraded
proteins, other organic compounds, or ions.

A related method used commonly in forensic laboratories utilizes Chelex ion exchange resin
that binds multivalent metal ions and is particularly useful in removing inhibitors from DNA.
It can be used with any type of sample, including whole blood, bloodstains, seminal stains,
buccal swabs, or hair. The only difference from the previous method is the presence of resin,
which binds the impurities from the solution, while DNA is being left in the solution. By
centrifuging the samples, the resin is brought to a pellet and separated.

Another method similar to Chelex relies on the use of paramagnetic beads with DNA binding
capacity. Samples are lysed and then the solid material is treated with Proteinase K. The lysates
are then applied to the beads. Resin is subsequently washed and DNA is eluted of it at 65°C,
magnetic beads are separated from the sample on a magnetic stand.
Other methods of DNA purification involve columns of various sorts, which are packed with
ion exchange, or silica based resins or matrices. Ion exchange columns are generally positively
charged to bind the negatively charged DNA; silica matrices are also charged and can also
retain DNA. In such applications DNA from the cellular lysates is expected to bind to the
column. These columns are then washed using salt solutions to remove unbound material.
Nucleic acid is then recovered by applying water or a neutral pH salt solution to break down
the resin-DNA bonding.

The use of columns allows increased throughput of samples, shorter time of isolation in
comparison to traditional solvent based extraction, increased yield of recovered DNA, and
improved quality of purified DNA.

In addition to columns and the previously described resins, there are also liquid resins that are
used. The principle is the same as for magnetic beads, but at the final step the samples have to
be spun to separate DNA from the resin.

All of these methods so far have dealt with simple, single samples. In some cases a sample
consists of a mixture of cells, for example sperm cells and non-sperm epithelial cells. This
extraction is based on differential properties of the two cell types. Sperm cells resist Proteinase
K lysis; therefore the non-sperm cells are lysed first in its presence. When the tube is
centrifuged, the solution contains epithelial DNA, while the pellet contains sperm cells. Sperm
cells are subsequently lysed by adding dithiothreitol or DTT with Proteinase K. Any of the
techniques mentioned before can be used to isolate the DNA from those differential lysates.

Although plants are not a common source of DNA for forensic investigation, analysis of their
DNA is very common in science. Plants are more difficult to work with than many other
materials for a couple of reasons. First, plant cells have a cell wall, which has to be at least
partly destroyed before the cytoplasm with the DNA can be accessed. Second, plants often
have high levels of sugars (for example starch or fructose) in their tissues or other organic
compounds such as polyphenols.

Grinding of the samples in liquid nitrogen helps to destroy the cell wall, but the organic
compounds including sugars still remain. As a result, methods were developed that use
chloroform-octanol mix, hexadecyltrimethylammonium bromide (CTAB) with high salt to
remove polysaccharides, and polyvinylpyrrolidone (PVP) to remove polyphenols.
All of these methods are successfully used in various laboratories and with various samples.
The methods have to be properly selected to optimize the yield and quality of the DNA
extracted.

DNA precipitation is achieved by adding high concentrations of salt to DNA-containing

solutions, as cations from salts such as ammonium acetate counteract repulsion caused by the
negative charge of the phosphate backbone. A mixture of DNA and salts in the presence of
solvents like ethanol (final concentrations of 70%–80%) or isopropanol (final concentrations
of 40%–50%) causes nucleic acids to precipitate. Some protocols include washing steps with
70% ethanol to remove excess salt from DNA. Finally, nucleic acids are re-suspended in water
or TE buffer (10 mM Tris, 1 mM ethylenediaminetetraacetic acid [EDTA]). TE buffer is
commonly used for long-term DNA storage because it prevents it from being damaged by
nucleases, inadequate pH, heavy metals, and oxidation by free radicals. Tris provides a safe pH
of 7–8, and EDTA chelates divalent ions used in nuclease activity and counteracts oxidative
damage from heavy metals.

References:

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Optimization of a semi-nested multiplex PCR to identify plasmodium parasites in wild-
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[6]Swain .S ., Mohanty .A., Tripathy .H.K., Mahapatra .N., Kar, S.K., Hazra,R.K., :
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