Field of the Invention:
The present invention relates to the field of nucleic acid extraction from the plant material.
Particularly, the invention provides a high yielding DNA extraction process from oilseed crops,
medicinal plants or plants containing high quantity of polyphenols, polysaccharide etc. The
extraction process essentially comprises use of EDTA as fixing agent and high concentration of
polyvinyl pyrrolidone and P mercaptoethanol in a particular ratiolamount which surprisingly
makes the process effective by increasing the yield to about 6390 pg/grn.
Said process is quick, simple, economical and an efficient process which is capable of generating
large amount of DNA with high purity by utilizing very less quantity of starting plant material.
In the present embodiment, the invention has been described with reference to oil seed crops,
however such description should not be considered as restricting the scope of the present
invention. Further it would be possible for a person skilled in the art to practice the present
invention considering other crops containing high quantity of polyphenol, polysaccharide etc. for
the extraction of DNA without departing from the scope of the present invention.
Background of the invention:
India has a wide range of oilseed crops grown in different agro climatic zones. Sesame, Sarson,
Taramira, Jatropa and Groundnut are traditionally cultivated oilseeds crops. Modern tools like
molecular techniques are being employed for enhancing the efficiency of resistance breeding to
make oil seed production competitive and sustainable. The extraction of DNA, RNA and protein
is required in molecular biology for the subsequent downstream processes, analytical, or
preparative purposes. The process of DNA isolation is a very crucial step for the molecular
analysis of any plant species and the process becomes even more difficult when the plant species
contain high amount of secondary metabolites like flavonoid, essential oil, phenolic acid,
polysaccharide etc. These compounds particularly in oilseed crops or medicinal plants are
considered to be as contaminants that causes degradation of DNA.
Depending on the nature and complexity of the plant materials, different methods have been
developed and modified by many laboratories for genomic DNA isolation (Murray and
Thompson, 1980; Taylor and Powell, 1982; Dellaporta et al., 1983; Doyle and Doyle, 1987;
Doyle and Doyle, 1990; Csaikl et al., 1998; Wulff et al., 2002; Sharma et al., 2008). Literature
survey for the last two decades clearly reveals that the cationic detergent, Cetyl Trimethyl
Ammonium Bromide (CTAI3)-based DNA isolation protocols were more frequently used for
different plant materials as compared to other protocols. Apart from cationic and/or anionic
detergents, different chemicals and biochemicals, such as sodium perchlorate, lysozyme,
PolyVinylPyrolidone (PVP), pectinases, proteinase K and others were included at different steps
for improving efficiency of the DNA isolation protocols. However, the yield of the DNA was not
found good and therefore research is still going on in the domain for obtaining a high yielding
extraction process.
Polyvinylpyrrolidone (PVP), a compound known to suppress polyphenolic oxidation, has been
used frequently in CTAB extraction method (Doyle and Doyle, 1990). The modified CTAB
buffer was also employed to extract DNA from T. minuta (a medicinal plant) using liquid
nitrogen (Hills and Van Staden, 2002). However, it is reported that there was no significant
increase in yield or in prevention of contamination of the DNA (Schneerman et al., 2002).
Further, the SDS-based extraction buffer is being used to break open the cells and isolate DNA
but the quality of DNA obtained is questioned due to precipitation of polysaccharides and
proteins.
The currently existing DNA extraction protocols are not suitable for oilseed crops because of the
presence of high level of polyphenols & essential oils therein. Various protocols such as CTAB
and salt extraction methods (Doyle and Doyle, 1987) have been described, but those tend to use
liquid nitrogen for crushing the plant material, which is quite expensive and require extensive
precautions and such methods does not yield high amount of DNA with good quality.
It has also been seen that a method that is effective in DNA extraction in one plant group often
fails when used on other plants. CTAB method has been seen useful in some plants but is not
considered successful for oilseed plants.
I
The prior art methods also involved use of phenol for the purification of DNA, which is seen to
be hazardous to the users. Also, the existing protocols require large quantities of tissue (in
grams) to be crush, which requires long time for plant growth.
Reference has been made to a non patented literature, "DNA extraction from olive oil and its use
in the identification of the production cultivar" by Matteo Busconi, Chiara Foroni, Massimiliano
Corradib, Cristina Bongiornia, Federica Cattapan, Corrado Fogher. The cited non patented
literature is known to use the CTAB method in as described in Doyle and Doyle, 1987.
Use of liquid nitrogen is stated here which is very expensive. It is difficult for small laboratories
to arrange for liquid nitrogen. The initial plant tissue amount taken is also very large. The
experimental procedure requires about 250 grams of the plant tissue to be used.
Reference is further made to a patented invention US 5530192, "Oilseed crops producing
valuable seeds having altered amino acid composition and fatty acid composition". The patented
invention discusses the use of liquid nitrogen, which is a very expensive ingredient. The plant
tissue used is also 5 grams. The plant needs to replenish the tissues and hence use of a large
quantity is not preferred in this method.
The DNA isolation method using liquid nitrogen from seeds of Brassica napus has been
disclosed in the art (known through Li Maoteng, Liu Jainmin, Zhangyi et al.)
In the same line, US 5,204,246 discloses a method for the isolation of DNA from plants, yeast
and bacteria using sodium/potassium ethyl xanthogenate and liquid nitrogen. The extraction
process disclosed in document is also not effective in producing high yield.
Further reference has been made to Dellaporta plant DNA extraction by Dellaporta et.al. which
discloses use of liquid nitrogen for breaking the cell wall of the plant tissue for further use.
Hence, there is a need of a DNA extraction process for oilseed crops, which can eliminate the
need of liquid nitrogen and which is efficient in producing substantially good yield of highly
pure DNA from a very less starting material.
Also, there is a requirement of a DNA extraction process for plants which on one hand should be
simple and efficient in terms of yield and purity of DNA and on the other hand should be
economical.
Object of the Invention:
A primary object of this invention is to overcome the disadvantagesldrawbacks of the known art
of process for the extraction of DNA.
Another object of this invention is to provide a DNA extraction process which is capable of
producing large quantity of DNA from small quantity of plant material.
Another object of this invention is to provide a DNA extraction process which yields highly
purified DNA.
Another object of this invention is to provide for a quick, simple, cheaper and efficient DNA
extraction process.
Another object of this invention is to provide an extraction composition useful for the extraction
of DNA from tissue of plant.
Another object of this invention is to provide a plant DNA extraction kit which produces high
quantity of DNA by utilizing very small amount of starting material.
These and other advantages of the present invention will become readily apparent from the
following detailed description read in conjunction with the accompanying drawings.
Summary of the invention:
The present invention relates to the field of nucleic acid extraction from the plant material.
Particularly, the invention provides a process of obtaining DNA from a tissue sample of plants,
said process essentially comprising steps:
a) treating said tissue in a fixing solution followed by the process of grinding, said fixing
solution comprising 0.1 M to 5M EDTA;
b) mixing the treated tissue of step (a) with an extraction composition wherein said composition
essentially comprising EDTA in an amount ranging from 70mM to 120mM, PVP in an amount
ranging from 3% to 6%, P mercaptoethanol in an amount ranging from 3% to 6%;
c) mixing the reagent of step (b) with a detergent followed by incubation at around 5 5 ' ~to 6 5 ' ~
and cooling in water bath for about 1 to 2hours;
d) treatment of resulting mixture of step (c) with chloroform and isoamyl alcohol followed by
centrifbgation and precipitation of the resulting solution to obtain DNA;
said process is characterized in that, it yields about 6390 pglgm DNA.
In an embodiment, the invention also provides an extraction composition useful for the
extraction of DNA from a plant tissue, said composition comprising:
a) EDTA in an amount ranging from 70mM to 120mM,
b) PVP in an amount ranging from 3% to 6%,
c) p mercaptoethanol in an amount ranging from 3% to 6%;
d) Detergent in an amount of about 1 %,
e) NaCl in an amount of about 1 OOrnM,
f) Tris base in an amount of about 1 OOmM
In another embodiment, the invention also provides a plant DNA extraction kit comprising:
a) a fixing solution comprising 0.1M to 5M EDTA;
b) an extraction composition, wherein said composition essentially comprising EDTA in an
amount ranging from 70rnM to 120mM, PVP in an amount ranging from 3% to 6%, P
mercaptoethanol in an amount ranging from 3% to 6%; detergent comprising sodium Dodecyl
sulfate or Sodium lauryl sulfate in an amount of about 1 %, NaCl in an amount of about 1 OOmM,
Tris base in an amount of about 1 OOmM.
said detection kit is characterized in that, it yields about 6390 pglgm DNA.
Brief description of the drawing:
Figure 1 illustrates the Electrophoretic pattern of extracted DNA from different oil seed crops
Lane-M= Lambda DNA Eco RI/Hind I11 double digested marker
Lane-1 = Sarson, Lane-2= Taramira, Lane-3= Sesarnum, Lane-4= Jatropa, Lane-5= Groundnut
Detailed description of the invention:
The following description is of exemplary embodiments only and is not intended to limit the
scope, applicability or configuration of the invention in any way. Rather, the following
description provides a convenient illustration for implementing exemplary embodiments of the
invention. Various changes to the described embodiments may be made in the function and
arrangement of the elements described without departing from the scope of the invention.
The present invention provides a high yielding and an efficient process for extraction of DNA
from oilseed crops, medicinal plants or plants containing high quantity of polyphenols,
polysaccharide, tannins etc.,
The term "oilseed crops" as used herein refers to crops which are used to obtain oils (fats) from
their seeds. As described above, any oilseed crops that can be used for extraction of oils (fats)
from their seeds may be used in the present invention. The examples of oilseed crops include
sarson, taramira, sesamwn, jatropa, groundnut, rapeseed, Tougoma (hima), Egoma, peanut, olive,
soybean, maize, flax, sunflowers, and oilpalm. Preferable oilseed crops are sarson, taramira,
sesamum, jatropa, groundnut, soybeans, flex, sunflower etc. in the present invention.
The process of obtaining DNA from a tissue sample of plants, said process essentially comprises
following steps:
a) treating said tissue in a fixing solution followed by the process of grinding, said fixing
solution comprising 0.1 M to 5M EDTA;
b) mixing the treated tissue of step (a) with an extraction composition wherein said composition
essentially comprising EDTA in an amount ranging from 70mM to 120mM, PVP in an amount
ranging from 3% to 6%, P mercaptoethanol in an amount ranging from 3% to 6%;
c) mixing the reagent of step (b) with a detergent followed by incubation at around 5 5 ' ~to 6 5 ' ~
and cooling in water bath for about 1 to 2hours;
d) treatment of resulting mixture of step (c) with chloroform and isoamyl alcohol followed by
centrifugation and precipitation of the resulting solution to obtain DNA;
The process surprisingly yields approximately maximum of 6935 pglgm DNA and an average
of 3222pglgm. The extraction process essentially comprises use of EDTA as fixing agent and
high concentration of polyvinyl pyrrolidone and P mercaptoethanol in a particular ratiolamount
which surprisingly and advantageously makes the process effective by increasing the yield to
about 6390 pglgm.
The process takes less time as it requires very small amount of about 0.2 gram to 0.5 gram of
starting plant material.
The DNA has high purity content as the A2601 ratio is present in the range of 1.37-2.0. The
process also produces DNA with A2601A 280r atio reaching upto 1.9 or -2 indicating the good
efficiency of the process to yield highly pure DNA.
The process basically employs 1% sodium dodecyl suphate (SDS) buffer, high concentration of
Polyvinyl pyrrolidone (PVP) and P-mercaptoethanol (instead of liquid N2), NaC1, fixing solution
like EDTA has been used in the extraction process.
The process in detail includes following steps:
1. 0.2 gram to 0.5 gram of plant tissue is treated with a fixing solution for about 30 minutes
followed by grinding the fixed tissue in chilled mortar pestle to fine paste in order to
break the cell walls to release the cellular constituents.The fixing solution is preferably an
Ethylene DiamineTetraAcetic acid solution having concentration in the range of 0.1M to
5M.
2. Transferring the grind tissue into a pre-chilled (with dry ice) centrihge tube and mixing
the same with a preheated extraction composition of about 5ml in 50 ml flask at
approximately 60°C in a water bath. The extraction composition comprises following
components:
a) EthyleneDiamineTetraAcetic acid (EDTA) in an approximate amount ranging from
70mM to 120rnM;
b) Polyvinylpyrrolidone (PVP) in an approximate amount ranging from 3% to 6%;
c) p mercaptoethanol in an approximate amount ranging from 3% to 6%;
d) Detergent in an approximate amount of about 1%;
e) NaCl in an approximate amount of about 1OOmM;
f) Tris base in an approximate amount of about 1 OOmM;
The detergent is selected from sodium Dodecyl sulphate or sodium lauryl sulphate.
The extraction buffer is maintained at a pH of about 8.5. It is important to add PVP and
p- mercaptoethanol just before use. The cell membranes must be disrupted so that the
DNA is released into the extraction buffer. The extraction composition essentially
comprises high concentration of EDTA along with PVP and p mercaptoethanol in such a
ratio or proportion which surprisingly causes the enhanced extraction of DNA from the
tissue.
The step of fixing the tissue prior to grinding and treatment of the grinded tissue with the
above described extraction composition produces surprising results in terms of high yield
of about 6390 pglgrn DNA with A260/ A280 ratio of reaching approximately 2.
3. Incubation of tubes at approximately 60" C in a water bath for 1 hour followed by mixing
the solution gently in between the incubation period for uniform mixing.
4. Post incubation, centrifuge tubes are taken out of water bath and is allowed to leave for
10 minutes for cooling at room temperature.
5. Further equal volume (equal to the current total volume of the contents of the tube) of
chlorofonn/isoamyl alcohol in a ratio of 24: 1 is added to the solution. The solution is
mixed thoroughly to form a complete emulsion until the two separate phases can not be
differentiated (i.e., no chloroform phase should form at the bottom of the tube until
centrifugation). This step is very crucial in the extraction process.
6. Centrifugation of the tubes at 12000 rpm for approximately10 minutes at around 4°C.
7. Transfer the supernatant solution from the top (aqueous) phase into a new autoclaved
centrifuge tube.
8. Discard the lower (chloroform) phase which contains cell debris.
9. The upper aqueous phase will be clear, though often colored.
10. Repeat 6,7,8 and 9 steps thrice or more depending on the requirement until a clear upper
aqueous phase is not obtained.
11. Now precipitate DNA by adding 5 M NaCl and 213 volume of chilled isopropanol. Leave
for 2 hours at -20" C or overnight for complete precipitation.
12. P 1000 i.e.1 ml pipette is used for obtaining DNA present in the solution as DNA is a
long, gentle molecule that is easily sheared when it passes through a narrow opening.
This will help in improving the quality of DNA. Spooling out of nucleic acids with a
glass rod is preferable.
13. Further, centrifuge the precipitated DNA at approximately 10000 rpm for about 10
minutes at 4°C in order to obtain the DNA pellet. In case of oil seed crops, DNA pellet is
yellowish in color instead of white.
14. The pellet is further washed with 70% ethanol. This step is repeated twice.
15. Optionally, centrifugation can be done for washing the DNA pellet with 70% ethanol
followed by air drying the DNA pellet at room temperature by leaving overnight for
better yield.
16. Resuspend the air dried pellet in around 200 pl Tris EDTA (TE) buffer. The TE buffer
comprises10 mM Tris-HC1, 1 mM EDTA and the buffer is maintained at around pH 8.0.
17. Checking the DNA quality by running on 0.8% agarose gel and quantity by taking
absorbance at 260 nrn and 280 nrn.
This process is highly advantageous in producing good quantity of DNA by utilizing very small
quantity of starting material i.e. tissue, leaves etc. of about 0.2 to 0.5 gram. The use of EDTA as
a fixing agent contributes in making the extraction process highly economical as it costs only 1.2
Rs. Igm.
The below mentioned table (Table 1) shows differentiating features of the present process of
DNA extraction with the other existing protocols in the art.
Sharma
et al,
2010
Not used
1.4M
lOOmM
Tris
20mM
CTAB-
2%
0.2%
1.5-
2.5%
Dharma
n &
Selvaraj
9
2009
Not used
1.4M
lOOmM
Tris
20mM
CTAB-
2%
2%
2%
Present
Invention
Used
1 OOmM
lOOrnM
Tris-base
-100mM
SDS-1%
4%
4%
Sharma
et al,
(2003)
Not
used
1.4M
1OOmM
Tris
20mM
CTAB-
2%
0.2%
-
S.No. Compone Doyle &
nts Doyle
protocol
(1987)
1. EDTA as Not used
a fixing
agent
2. NaCl 1.4M
3. Tris-Hcl 1OOmM
/Tris-base Tris-Hcl
4. EDTA (in 20mM
Extraction
compositi
on)
Detergent CTAB-
2%
P-ME .2%
PVP -
Dellapo
rta,
(1983)
Not used
1OOmM
50mM
Tris-Hcl
1OmM
SDS-1%
Further, following table (Table 2) shows the efficacy of the present invention in terms of high
yield and purity of DNA. The table shows comparison of DNA yield of the present invention (in
different plants) and other DNA extraction process present in the domain.
Table 3, given below indicates the average yield and A2601 A 280 of DNA produced from the
present process.
S.No.
1.
2.
3.
4.
Process
Sharma et al, (2003)
Dharman &
Selvaraj, 2009
Sharma et al, 201 0
Present Invention
Plant
Sarson
Tararnira
Sesarnurn
Yield (pglgm)
Approximately 3 72
(Maximum)
Approximately 2710
(Maximum)
Approximately 2608
(Maximum)
Approximately 6390
(Maximum)
Plant Yield
Taramira 6390
Sarson 5750
Groundnut 4660
Jatropa 3875
Sesame 3625
A260
0.461
0.51 1
0.029
A 280
0.227
0.257
0.014
Yield
A2601 A 280 (pglg tissue)
2.03 5750
1.98 6390
2.0 3625
The above table exhibits that plant tissue of 1 gm produces about 3875 ug of DNA.
Jatropa
Groundnut
The invention in another aspect provides an extraction composition usehl for the
extractiodisolation of DNA from plant tissue. The composition comprises following
components:
a) EDTA in an amount ranging from 70mM to 120mM,
0.310
0.932
b) PVP in an amount ranging from 3% to 6%,
c) fl mercaptoethanol in an amount ranging from 3% to 6%;
0.152
0.504
d) Detergent in an amount of about 1 %,
2.03 3875
1.85 4660
e) NaCl in an amount of about 1 OOmM,
f) Tris base in an amount of about 1 OOmM
The detergent is selected from a group comprising of Sodium dodecyl sulfate or Sodium lauryl
sulfate. The plant is selected from a group comprising of oilseed plants, medicinal plants and
like.
In another aspect of the present invention, there is provided a plant DNA extraction kit. The kit
essentially comprises following components:
a) a fixing solution;
b) an extraction composition,
The fixing solution is EDTA or other reagentfchemical with similar chemical properties. The
EDTA is present in the molar range of 0.1M to 5M. The extraction composition essentially
comprises EDTA in an amount ranging from 70mM to 120mM, PVP in an amount ranging from
3% to 6%, Q mercaptoethanol in an amount ranging from 3% to 6%; detergent in an amount of
about 1%, NaCl in an amount of about 1 OOmM and Tris base in an amount of about 100mM. The
detergent comprises sodium Dodecyl sulfate or Sodium lawyl sulfate.
The detection kit is characterized in that it yields about 6390 pglgm DNA by utilizing small
quantity of DNA of about 0.2 gram.
The following description is of exemplary embodiments only and is not intended to limit the
scope, applicability or configuration of the invention in any way. Rather, the following
description provides a convenient illustration for implementing exemplary embodiments of the
invention. Various changes to the described embodiments may be made in the function and
arrangement of the elements described without departing from the scope of the invention.
In an embodiment of the present invention there is provided a process of obtaining DNA fiom
tissue sample of oil seed plants.
In another embodiment of the present invention there is provided a high yielding DNA extraction
process producing upto 6390 pg/gm DNA.
In another embodiment of the present invention there is provided a DNA extraction process
which produces DNA with Az6()/ A280 ratio of approximately 1.9 or 2.
In another embodiment of the present invention there is provided an extraction composition for
the extraction of DNA fiom plant tissue.
In another embodiment of the present invention there is provided a plant DNA extraction kit
comprising a fixing solution and extraction composition.
The following are examples are provided solely to illustrate the present invention and are not
intended to limit the scope of the invention, as described herein.
Example 1:
About 0.2 g of plant tissue was taken which was further fixed by treating it with 1M EDTA for
about 30 minutes. Then, the fixed tissue was then ground in chilled mortar pestle to fine paste.
The cell walls must be broken (or digested away) in order to release the cellular constituents. The
ground tissue was then transferred into a pre-chilled (with dry ice) centrifuge tube.
The tissue was then treated with extraction composition comprising the mixture of 90mM
EDTA, 4% PVP and 5% P mercaptoethanol. Further, the treated tissue is mixed with SDS
and was further incubated at 60 degree celsius and cooled in water bath for about 1 hour. The
resulting mixture was treated with chloroform and isoarnyl alcohol followed by
centrifugation and precipitation to obtain the extracted DNA. The process yielded about
3022pglgm of DNA.
Example 2:
About 0.3 g of plant tissue was taken which was further fixed by treating it with 2M EDTA
for about 30 minutes. Then fixed tissue was then ground in chilled mortar pestle to fine paste.
The cell walls must be broken (or digested away) in order to release the cellular constituents.
The grind tissue was then transferred into a pre-chilled (with dry ice) centrifuge tube. The
tissue was then treated with extraction composition comprising the mixture of 80rnM EDTA,
5% PVP and 3% P mercaptoethanol. Further, the treated tissue is mixed with SDS and was
hrther incubated at 65 degree celsius and cooled in water bath for about 1.5 hour. The
resulting mixture was treated with chloroform and isoarnyl alcohol followed by
centrifugation and precipitation to obtain the extracted DNA. The process yielded about
2749pglgm of DNA.
Example 3:
About 0.3 g of plant tissue was taken which was fbrther fixed by treating it with 3M EDTA
for about 30 minutes. Then fixed tissue was then ground in chilled mortar pestle to fine paste.
The cell walls must be broken (or digested away) in order to release the cellular constituents.
The grind tissue was then transferred into a pre-chilled (with dry ice) centrifuge tube. The
tissue was then treated with extraction composition comprising the mixture of lOOmM
EDTA, 4% PVP and 5% j3 mercaptoethanol. Further, the treated tissue is mixed with SDS
and was hrther incubated at 55 degree celsius and cooled in water bath for about 2 hour. The
resulting mixture was treated with chloroform and isoarnyl alcohol followed by
centrifugation and precipitation to obtain the extracted DNA. The process yielded about
1792pglgm of DNA.
Example 4:
About 0.2 g of plant tissue was taken which was further fixed by treating it with 0.5M EDTA
for about 30 minutes. Then fixed tissue was then ground in chilled mortar pestle to fine paste.
The cell walls must be broken (or digested away) in order to release the cellular constituents.
The grind tissue was then transferred into a pre-chilled (with dry ice) centrifuge tube. The
tissue was then treated with extraction composition comprising the mixture of 1% SDS,
lOOrnM NaCl, lOOrnM EDTA, 4% PVP and 4% P mercaptoethanol. Further, the treated
tissue is incubated at 60 degree celsius for 1 hour and cooled at room temperature for about
10 min. The resulting mixture was treated with chloroform and isoarnyl alcohol followed by
centrifugation and precipitation to obtain the extracted DNA. The process yielded about 6390
pg/gm of DNA.
Advantages of the present invention:
The process is comparatively quicker, cheaper and consistent for extraction of high quality
DNA from oil seed crops.
The process produces high yield of DNA from small amount of plant material. On an
average of 4094.2 pg/gm of DNA with maximum of 6390 pglgm and minimum of 362
pg/gm tissue.
The process eliminates the necessity of phenol or any other toxic chemicals, which makes
the method less hazardous. Thus, the process is quite safe and cost effective.

WE CLAIM
1. A process for obtaining DNA from a plant tissue sample, said process essentially comprising
steps:
a) treating said tissue sample with a fixing solution followed by the grinding process wherein
said fixing solution comprises 0.1M to 5M EDTA;
b) mixing treated tissue resulting from step (a) with an extraction composition wherein said
extraction composition essentially comprises EDTA in an amount ranging from 70mM to
120mM, PVP in an amount ranging from 3% to 6%, P mercaptoethanol in an amount ranging
from 3% to 6%;
c) mixing the solution resulting from step (b) with a detergent followed by incubation at a
temperature ranging from 5 5 ' ~to 6 5 ' ~a nd cooling the resulting solution in a water bath for
about 1 to 2 hours;
d) treating solution resulting from step (c) with reagent(s) selected from a group comprising
chloroform, isoarnyl alcohol, followed by centrifugation and precipitation of the resulting
solution to obtain DNA;
said process is characterized in that it yields to about 6390 pg DNA per gram tissue sample.
2. The process as claimed in claim 1, wherein said DNA has a A260/ A280 ratio of approximately
3. The process as claimed in claim 1, wherein said detergent is selected from a group comprising
of Sodium dodecyl sulfate, Sodiurn lawyl sulfate.
4. The process as claimed in claim 1, wherein said extraction composition further comprising
Sodium dodecyl sulfate in an amount of about 1%, NaCl in an amount of about 100mM, Tris
base in an amount of about 100mM.
5. The process as claimed in claim 1, wherein said tissue sample is selected from leaves. stems,
and roots.
6. The process as claimed in claim 1, wherein said plant is selected from a group comprising
oilseed plants, medicinal plants and like.
7. The process as claimed in claim 1, wherein said process utilizes amount of said plant tissue as
low as about 0.2gram.
8. An extraction composition for extraction of DNA from plant tissue sample, said extraction
composition comprising:
a) EDTA in an amount ranging fiom 70mM to 120mM
b) PVP in an amount ranging from 3% to 6%
c) p mercaptoethanol in an amount ranging from 3% to 6%
d) Detergent in an amount of about 1 %
e) NaCl in an amount of about 1 OOmM
f) Tris base in an amount of about 100mM
9. The extraction composition as claimed in claim 8, wherein said detergent is selected fiom a
group comprising Sodium dodecyl sulfate, Sodium lauryl sulfate.
10. The extraction composition as claimed in claim 8, wherein said plant is selected from a group
comprising oilseed plants, medicinal plants and like.
11. A plant DNA extraction kit comprising:
a) a fixing solution comprising 0.1M to SM EDTA;
b) an extraction composition comprising EDTA in an amount ranging fiom 70mM to 120mM,
PVP in an amount ranging from 3% to 6%, Q mercaptoethanol in an amount ranging from 3% to
6%, a detergent selected from a group comprising sodium dodecyl sulfate or Sodium lauryl
sulfate in an amount of about 1%, NaCl in an amount of about 100mM, Tris base in an amount
of about 100mM;
c) a plant tissue sample;
12. The extraction kit as claimed in claim 11, wherein said detection kit yields to about 6390pg
DNA per gram tissue sample.
13. The extraction kit as claimed in claim 1 1, wherein said kit utilizes amount of said plant tissue
as low as about 0.2gram.
14. The extraction kit as claimed in claim 11, wherein said plant is selected from a group
comprising oilseed plants, medicinal plants and like.