CLIAMS:1. A method of isolating DNA from a food sample, said method comprising the steps of:
a. subjecting said food sample to a protein denaturation treatment to obtain a denatured food sample;
b. defatting said denatured food sample to obtain a defatted sample;
c. contacting said defatted sample with 50-90% ethyl alcohol, and obtaining said defatted sample as dry solids after removing ethyl alcohol by centrifugation;
d. grinding said dry solids; and
e. isolating DNA from said dry solids using conventional DNA isolation methods.
2. The method of isolating DNA from a food sample as claimed in claim 1, wherein said food sample is optionally grinded before subjecting to said protein denaturation treatment.
3. The method of isolating DNA from a food sample as claimed in any of the claims 1-2, wherein said protein denaturation treatment is a high temperature treatment at 55-650C for 20-30 minutes.
4. The method of isolating DNA from a food sample as claimed in any of the claims 1-3, wherein said food sample is defatted by mixing said food sample with water maintained at 60 to 700C and followed by removing fat by centrifugation.
5. The method of isolating DNA from a food sample as claimed in any of the claims 1-4, wherein said ethyl alcohol is removed by centrifuging said defatted sample at 6000 to 7000 rpm for 15 to 20 minutes and completely draining the liquid to obtain said dry solids.
6. The method of isolating DNA from a food sample as claimed in any of the claims 1-5, wherein said dry solids are grinded using liquid nitrogen.
7. The method of isolating DNA from a food sample as claimed in any of the claims 1-6, wherein said conventional DNA isolation method comprises the steps of:
a. contacting said dry solids with a preheated DNA extraction buffer and homogenizing to obtain a solution;
b. holding solution of step a) at 50- 650C for 15-20 minutes;
c. placing solution of step b) on ice and contacting said solution with equal volume of chloroform: isoamyl alcohol (24:1) and mixing well;
d. carrying out a centrifugation step at 6000-8000 rpm for 20-30 minutes at 40C to obtain a lower organic layer and an upper aqueous layer and collecting said aqueous layer in a fresh tube;
e. adding half the volume of pre-chilled 5M sodium chloride to aqueous layer of step d), mixing well and then adding equal volume of pre-chilled isopropanol and holding at -200C for 30-60 minutes;
f. carrying out a centrifugation step at 12000-13000 rpm for 25-30 minutes to obtain a pellet and a supernatant, and discarding said supernatant by keeping the tubes in inverted position on a sterile paper towel;
g. gently washing pellet of step f) with 70% ethanol and discarding said ethanol;
h. drying pellet of step g) at 55-600C in for 20 minutes;
i. contacting pellet of step h) with Tris-EDTA (TE) buffer to obtain a crude DNA solution;
j. contacting crude DNA solution of step i) with RNAse A and holding at 370C for 60 minutes;
k. adding equal amount of chloroform: isoamyl alcohol (24:1) to crude DNA solution of step j) and mixing well;
l. carrying out a centrifugation step at 11000-13000 rpm for 15-20 minutes to obtain a lower organic layer and a upper aqueous layer;
m. contacting aqueous layer of step l) with proteinase K and holding at 370C for 60 minutes;
n. adding equal amount of chloroform: isoamyl alcohol (24:1) to aqueous layer of step m) and mixing well;
o. carrying out a centrifugation step at 11000-13000 rpm for 15-20 minutes to obtain a lower organic layer and a upper aqueous layer;
p. adding double the volume of absolute ethanol to aqueous layer of step o) and holding at 40C for 60 minutes;
q. carrying out a centrifugation step at 11000-13000 rpm for 15-20 minutes to obtain a pellet and a supernatant; and
r. contacting pellet of step q) with Tris-EDTA (TE) buffer.
8. A method as claimed in any of the claims 1-7 for use in determining genetic identity of plant biological material in a food sample.
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