Description:FIELD OF THE INVENTION

[001] The present invention relates to a method and composition and kit for rapid extraction of DNA. In particular, the present invention relates to method of rapid DNA extraction, composition and kit for DNA extraction from biological sample, specifically needle prick blood sample.

BACKGROUND OF THE INVENTION

[002] Molecular method are more sensitive and widely used method for clinical diagnostics. DNA extraction is the first step in such DNA-detection based diagnostic test. Currently commercially available DNA extraction kits require trained manpower, resources and high-end equipment and therefore can be performed only in laboratories. They are costly also.

[003] Moreover, blood collection for any diagnostic test is biggest logistic issue with the large animal as bringing animal to clinic or sending phlebotomist for blood collection to the animal (at site) is difficult and costly which delay diagnosis and finally treatment.

[004] Thus there is a long felt need for a user friendly, rapid, cost effective, less time-consuming method and composition and kit for DNA extraction from biological sample such as blood or cell culture.

OBJECTS OF THE INVENTION

[005] It is an object of the present invention, to provide a method and composition and kit for rapid DNA extraction.

[006] It is another object of the present invention, to provide method and composition and kit for rapid DNA extraction from biological sample, specifically needle prick whole blood sample.

[007] It is yet another object of the present invention, to provide method and composition and kit for DNA extraction that is membrane based.

[008] It is another object of the present invention, to provide method and composition and kit that eliminates the need of high-end equipment.

[009] It is another object of the present invention, to provide a method and composition and kit that enables storage of blood sample for 48 hours at room temperature therefore easy to transport samples from field to clinic or centralized laboratory.

SUMMARY OF THE INVENTION

[010] The present invention provides method and composition and kit for rapid DNA extraction from a biological sample. The said method and composition and kit enables easy storage of blood sample at room temperature for 48 hours.

[011] The present invention further discloses a method and composition and kit that enables DNA exaction from biological sample without utilizing large equipment. Furthermore, the said method and composition and kit for DNA extraction is equipment free, membrane based and cost effective. Moreover, the said method cam readily be performed at, but not limited to, the site of sample collection, in the field or at low resource requiring clinics.

[012] To further clarify advantages and features of the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which is illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings and examples.

DETAILED DESCRIPTION OF THE INVENTION

[013] The embodiments of the invention will now be described herein, with reference to the accompanying examples and drawings. It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.

[014] The present invention provides a method and composition and kit for rapid DNA extraction from a biological sample, such as but not limited to needle prick whole blood sample, cell culture etc, in 10 - 15 minutes. Further, the invention enables DNA exaction from biological sample without utilizing large equipment and is paper based and cost effective. Furthermore, the said method and composition and kit enables easy storage of DNA for 48 hours even at room temperature. Moreover, the said method cam readily be performed at, but not limited to, the site of sample collection, in the field or at low resource requiring clinics.

[015] According to an embodiment of the present invention, the composition comprises a L1 buffer and Triton X-100, wherein the Triton X-100 is 0.8-1.5%.

[016] According to another embodiment of the present invention, the L1 buffer comprises 2-5mM MgCl2, 0.4-0.8% DMSO, 5-8% SDS and 12-16 mM Tris-HCl, pH 8.2.

[017] According to yet another embodiment of the present invention, ratio of L1 buffer and Triton X-100 in the composition is 4.4:1 to obtain the lysis buffer.

[018] According to an embodiment of the present invention, the method of extracting DNA from a sample includes the steps of:

(a) dropping biological sample onto membrane;
(b) dropping lysis buffer onto the sample loaded membrane;
(c) washing the membrane with wash buffer;
(d) allowing washed membrane to dry; and
(e) eluting DNA with elution buffer.

[019] According to an embodiment of the present invention, the biological sample utilized for DNA extraction or isolation is 35 µL – 150 µL on a membrane of 5-9 mm diameter. The buffers utilized for DNA extraction are 150-250 µL lysis buffer, 650-850 µL wash buffer and 130-160 µL elution buffer. The membrane is dried for 2 – 5 minutes at room temperature.

[020] According to an embodiment of the present invention, a DNA extraction kit to extract DNA rapidly from biological sample is provided. The DNA extraction kit includes extraction cassette and in discrete containers reagents to extract the DNA.

[021] According to another embodiment of the present invention, the reagents in discrete containers includes L1 buffer, 0.8-1.5% Triton X-100, W1 buffer and E1 buffer. The W1 buffer is wash buffer comprising 8-12 mM NaOH and E1 buffer is elution buffer comprising of 12-16 mM Tris-HCL, 0.05-0.5 mM EDTA pH 8.5.

[022] According to yet another embodiment of the present invention, the extraction cassette comprises 4 – 5 blotting sheets arranged one onto another and fusion 5 membrane. The blotting sheets are 2.5 cm x 2.8 cm x 0.03 cm and the membrane is 5 - 9 mm in diameter. The membrane is arranged in the center on top of the blotting sheets. Further, the membrane and blotting sheets are cased in a cover. The cover comprises a top and a bottom. Further, the cover of the cover is made of a material that is non-reactive towards the reagents and DNA such as, but not limited to, plastic, food grade paper and fine cardboard.
[023] According to another embodiment of the present invention, the biological sample includes mammalian cells or bacterial cells. Further, the mammalian cells include needle prick blood cells or stored blood cells.

[024] According to an embodiment of the present invention, the DNA extraction or isolation utilizing the said method is completed in 10 - 15 minutes.

[025] According to another embodiment of the present invention, the DNA is extracted without the use of any large instruments making the method user friendly and cost effective. The DNA in the biological sample or whole blood remains intact on the membrane upto 48 hours at room temperature (with no refrigeration required) making the transportation of DNA easy and safe. The said DNA is PCR/LAMP/RT-PCR compatible.

[026] According to yet another embodiment of the present invention, the method and composition and kit are highly sensitive and absorbs minimum debris protein.

EXAMPLES
[027] According to another embodiment of the present invention, the DNA was extracted from bovine needle prick blood sample utilizing the method as described above. The results showed that the DNA extracted successfully.

[028] According to yet another embodiment of the present invention, the DNA extraction was done. Firstly, 35-100 µl of fresh blood sample (drop by drop until it gets absorbed completely) was added by dropper to the membrane of prepared cassette. Then 150-250 µl lysis buffer was added to the membrane and subsequently the membrane was washed with 650-850 µl of wash buffer in a dropwise manner. Next the membrane was dried for 2 – 5 minutes. 150 µl elution buffer was added to an eppendorf and dried membrane was placed in eppendorf containing elution buffer. The membrane was incubated for 2 – 5 minutes and subsequently DNA was eluted by vortexing for 1 – 2 minute. The DNA extracted was quantified using nanodrop and stored at -20°C for future use.

[029] According to an embodiment of the present invention, the DNA extraction was done by loading sample onto membrane by contact with animal. Firstly, fresh blood sample (drop by drop until it gets absorbed completely) was added by pricking needle on animal to the membrane of prepared cassette. Then 150-250 µl lysis buffer was added to the membrane and subsequently the membrane was washed with 650-850 µl of wash buffer in a dropwise manner. Next the membrane was dried for 2-5 minutes. 130-160 µl elution buffer was added to an eppendorf and dried membrane was placed in eppendorf containing elution buffer. The membrane was incubated for 2-5 minutes and subsequently DNA was eluted by vortexing for 1-2 minute. The DNA extracted was quantified using nanodrop and stored at -20°C for future use.

[030] It will be apparent to those skilled in the art that various modifications and variations can be made in the products and methods of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Additionally, the foregoing examples are appended for the purpose of illustrating the claimed invention, and should not be construed so as to limit the scope of the claimed invention.
, C , C , Claims:We claim:

1. A composition, comprising L1 buffer and Triton X-100, wherein the Triton X-100 is 0.8-1.5%.

2. The composition as claimed in claim 1, wherein the ratio of L1 buffer and Triton X-100 is 4.4:1.

3. The composition as claimed in claim 1, wherein the L1 buffer comprises 2-5 mM MgCl2, 0.4-0.8% DMSO, 5-8% SDS and 12-16 mM Tris-HCl, pH 8.2.

4. A method of extracting DNA from a biological sample, the method comprising the steps of:

(a) dropping biological sample onto membrane;
(b) dropping lysis buffer onto the sample loaded membrane;
(c) washing the membrane with wash buffer;
(d) allowing washed membrane to dry; and
(b) eluting DNA with elution buffer.

5. The method as claimed in claim 4, wherein the amount of biological sample is 35 µL – 150 µL on a membrane of 5-9 mm diameter.

6. The method as claimed in claim 4, wherein the ratio of lysis buffer, wash buffer and elution buffer is 2:8:1.5.

7. The method as claimed in claim 4, wherein the membrane is dried for 2 – 5 minutes at room temperature.

8. The method as claimed in claim 4, wherein the biological sample includes mammalian cells or bacterial cells.

9. The method as claimed in claim 4, wherein the mammalian cells include needle prick blood cells or stored blood cells.

10. A DNA extraction kit to extract DNA rapidly comprising:

(a) extraction cassette; and
(b) reagents to extract DNA.

11. The DNA extraction kit as claimed in claim 8, wherein the reagents comprise L1 buffer, 0.8-1.5% Triton X-100, W1 buffer and E1 buffer.

12. The DNA extraction kit as claimed in claim 8, wherein the W1 buffer comprises 8-12 mM NaOH and E1 buffer comprises 12-16mM Tris-HCL, 0.05-0.5 mM EDTA pH 8.5.

13. The DNA extraction kit as claimed in claim 8, wherein the extraction cassette comprises 4 – 5 blotting sheets arranged one onto another and fusion 5 membrane.

14. The DNA extraction kit as claimed in claim 8, wherein in the extraction cassette the membrane is arranged in the center on top of the blotting sheets.

15. The DNA extraction kit as claimed in claim 8, wherein the membrane and blotting sheets are cased in a cover.

16. The DNA extraction kit as claimed in claim 8, wherein the cover comprises a top and a bottom.

17. The DNA extraction kit as claimed in claim 8, wherein the cover is made of plastic, food grade paper or fine cardboard.