Practical No : 11

Practical : Isolation of DNA extraction from plant tissue

Objectives : To practice the steps involving DNA extraction from plants. To understand the

importance of chemicals and conditions used in the experiment.

Introduction :

For the characterization of proteins, the protein has to be extracted from the living
tissue. Proteins from frozen tissue samples need to be extracted efficiently and without degradation
to make the best use of a limited resource and to ensure as much as possible, that an accurate
representation of the proteins in the living tissue is obtained. Different methods are used for different
tissues depending on the type of the tissue as well as type of protein that is going to be extracted.

A) Protein extraction from seeds

Plant tissues contain a wide range of proteins, which vary gently in their properties, and require
specific conditions for their extraction and purification. Plant tissues do pose specific problems,
which must be taken into account in extraction of proteins. The first is the present of rigid cellulose
cell wall, which must be shared to release the cell contents. Breaking up fresh tissue can be achieved
with grinding mortar and pestle or adding liquid nitrogen to rapidly freeze the material before
blending. The second is the presence of protein degrading and modifying compounds, such as
phenolic compounds and proteinases, which should be eliminated. Proteins whose activities are
sensitive to oxidation require extraction in the presence of reducing agents such as dithiothreitol
(DTT) or merchaptoethanol.

Materials : Mortar and pestle

Plant tissues (soaked seeds samples)

UV/VIS Spectrophotometer

SDS Extraction buffer (0.175 M Tris-HCl pH 8.8, 5% SDS, 15% Glycerol, 0.3 M DTT)

100% acetone

0.05 M Tris-HCl buffer pH 8.0

Methods :

First 1 g of fresh seeds were ground to a powder with mortar and pestle. Then
5 ml of SDS extraction buffer was added directly to motar and continue grinding for an additional
30s. Then the homogenate was centrifuged at 5000 rpm for 5 min and was collected the supernatant
 into a 50 ml tube. After that immediately four volumes of ice cold 100% acetone were added to
recovered supernatant. Then the final protein precipitate was collected by centrifugation at 5000 rpm
for 15 min and the pellet was dried. Finally the pellet was re-suspended in 1ml 0.05 M Tris-HCl
buffer pH 8.0.

Observations

Eppendorf

Extracted DNA from seed samples

Discussion
The basic steps used for DNA isolation, as mentioned above, need to be modified to consider the
different characteristics of the plant tissue and cells. Chemicals or enzymes used to lyse microbial
cells may not be equally effective on plant cells, for example, lysozyme is often included in kits to
lyse bacterial cells but has no effect on plant cells. Furthermore, the biochemical content within plant
cells is very different from microorganisms and animal cells. Many plant species have high content
of polysaccharides and polyphenols which are not removed by phenol extraction (unlike microbes).
Methods different from those used for microbes need to be included in kits to address the special
features of plant cells.

One method is to utilize a detergent called cetyltrimethylammonium bromide (CTAB) which forms
an insoluble complex with nucleic acid and selectively precipitates DNA, leaving behind
carbohydrates, proteins and other contaminating components. The DNA-containing precipitate can
be decomplexed by dissolving it in 1N NaCl. CTAB can be included in any step of the extraction
process. To remove polyphenols higher concentration of CTAB with polyvinylpyrrolidone (PVP) or
polyvinylpolypyrrolidone (PVPP) can be employed.

Another method is to use guanidium thiocynate (GITC), which assists DNA purification from plant
materials in two ways. Firstly, it denatures and dissolves proteins, disintegrates cellular structures,
and dissociates nucleoproteins from nucleic acid. Due to this property, GITC can be used to release
DNA from almost any type of tissue. Secondly, DNA binds strongly to silica particles in the
presence of GITC. This property can be utilized to separate DNA from the denatured proteins and
other biochemicals or cellular components. Commonly silica particles are packed in chromatography
columns and DNA extract treated with GITC is applied to it. DNA binds selectively to the column
and can be eluted in the last step after washing away the cellular contaminants.

In some DNA extraction procedures ascorbic acid, diethyldithiocarbamic acid and 2-

mercaptoethanol might be included to protect DNA against oxidation and degradation. RNA can be
removed by using RNase. The quality of the DNA isolated is dependent to a larger extent on the
physiological condition of the plant material rather than the kit protocol. Isolation of plant nucleic
acids for use for Southern blot analysis, polymerase chain reaction (PCR) amplifications, restriction
fragment length polymorphisms (RFLPs), arbitrary primed DNA amplifications (RAPD,
APPCR,DAF), and genomic library construction is one of the most important and time-consuming
steps. The degree of purity and quantity varies between applications. A good extraction procedure for
the isolation of DNA should yield adequate and intact DNA of reasonable purity. The procedure
should also be quick, simple and cheap and, if possible, avoid the use of dangerous chemicals.

The extraction process involves, first of all, breaking or digesting away cell walls in order to release
the cellular constituents. This is followed by disruption of the cell membranes to release the DNA
into the extraction buffer. This is normally achieved by using detergents such as sodium dodecyl
sulphate (SDS) or cetyl-methylammonium bromide (CTAB). The released DNA should be protected
from endogenous nuclease. EDTA is often included in the extraction buffer to chelate magnesium
ions, a necessary co-factor for nucleases, for this purpose. The initial DNA extracts often contain a
large amount of RNA, proteins, polysaccharides, tannins and pigments which may interfere with the
extracted DNA and difficult to separate. Most proteins are removed by denaturation and
precipitation from the extract using chloroform and/or phenol. RNAs on the other hand are normally
removed by treatment of the extract with heat treated RNase A.

Mechanical lysis for DNA extraction

Lysis can be done either by mechanically or chemically. In this practical we used mechanical lysis
process. Mechanical lysis means disruption of cells using sonication, a pressure cell, homogenizer, or
bead beater. Mechanical lysis methods are economical and preferable for large-scale preparations
because the addition of chemicals is not required. However, mechanical lysis produces heat, which
needs to be controlled. Care should also be taken to avoid foaming, to prevent surface denaturation
and oxidation. In our practical Cell lysis was performed by hand, using mortar and pestle.

While the grinding the lysosomes in the cells will also be broken and the digestive enzymes
including DNases in them will be released to the solution, which may degrade the DNAs prior to the
extraction. To avoid this the sample was frozen or added with liquid nitrogen, which provides a low
temperature environment, where the DNases are inactive.

As important as DNA molecules are to life, they are still extremely fragile, and break apart easily
when removed from cells. To slow down the rate at which the DNA breaks up, we cool down the
buffer solution to near freezing. Chemical reactions always take place slower in cold solutions than
in warm ones, because there is a lot less energy around to make the reaction take place.

Figure 01: Different DNA extraction methods from plant parts

CTAB (Cetyl trimethylammonium bromide)

Polysaccharide-like contaminants are more difficult to remove. They can inhibit the
activity of certain DNA-modifying enzymes and may also interfere in the quantification of nucleic
acids by spectrophotometric methods .NaCl at concentrations of more than 0.5 M, together with
CTAB is known to remove polysaccharides. CTAB will combine with the polysaccharides and
proteins and chemically alter them. CTAB binds to the cell wall debris, proteins and polysaccharides
in the homogenate: once perform a phenol-chloroform extraction after the CTAB step, the
CTAB/debris/polysaccharide/protein mix is removed.
Figure 02: Chemical structure of CTAB

In Plant DNA isolation due to its high polyphenolic content, this may interfere with the DNA purity
especially for subsequent manipulations. Antioxidants are commonly used to deal with problems
related to phenolics such as 2 -mercaptoethanol, ascorbic acid, Bovine Serum Albumin, sodium azide
and PVP amongst others. Phenol extractions when coupled with SDS are also helpful.

Buffer Solution

TE buffer is a commonly used buffer solution in molecular biology, especially in procedures

involving DNA or RNA. "TE" is derived from its components: Tris, a common pH buffer, and
EDTA, a molecule chelating cations like Mg2+. The purpose of TE buffer is to protect DNA or RNA
from degradation.

EDTA
Ethylenediaminetetraacetic acid, widely abbreviated as EDTA is a polyamino carboxylic acid and a
colourless, water-soluble solid. Its conjugate base is named ethylenediaminetetraacetate. It is widely
used to dissolve limescale. Its usefulness arises because of its role as a hexadentate ("si ligand and
chelating agent, i.e. its ability to "sequester" metal ions such as Ca2+ and Fe3+. After being bound by
EDTA, metal ions remain in solution but exhibit diminished reactivity.
 Figure 03: Atomic arrangement of Metal-EDTA complex

TrisHCl/EDTA buffer has 2 main functions, which includes the pH stabilization done by TrisHCl
and EDTA is a chelating agent and has great affinity with matel ions and Mg-ion present in DNase as
a cofactor and responsible for DNase action that degrade the DNA, here EDTA bind with Mg-ion
and nullify the action of DNase.

β-Mercaptoethanol
2-Mercaptoethanol is often included in extraction buffers designed for plant DNA extraction,
because it is a strong reducing agent which can remove tannins and other polyphenols often present
in the crude plant extract. It may also help to denature proteins by breaking disulphide bonds
between cysteine residues.

Phenol/Chloroform
The cell wall, some carbohydrates, lipids and proteins associated with the disrupted cell wall dissolve
in the phenol/chloroform. DNA is hydrophilic so remains in the aqueous portion.

Reference
1. Doyle J.J., Doyle J.L. (1990). Isolation of plant DNA from fresh tissue. Focus 12: 13-15.
2. Jobes D.V., Hurley D.L., Thien L.B. (1995). Plant DNA isolation: a method to efficiently remove
polyphenolics, polysaccharides, and RNA. Taxon 44: 349-386.
3. Sharma K.K., Lavanya M., Anjaiah V. (2000). A method for isolation and purification of peanut
genomic DNA suitable for analytical applications. Plant Mol. Biol. Rep. 18: 393a-393h.