Title of Invention

METHOD OF ISOLATION OF GENOMIC DNA USING UNCOATED MAGNETIC NANOPARTICLES

Abstract A method of isolation of genomic DNA using magnetic nanoparticles as solid phase support directly from the crude sample, or after preparing cell lysate and cleared cell lysate thereon, is disclosed. Additionally, the present invention also discloses the extraction of DNA fragment from agarose gel using magnetic nanoparticles as solid phase support.
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THE PATENTS ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION:
"ISOLATION OF GENOMIC DNA USING MAGNETIC NANOPARTICLES"
2. APPLICANT:
(a) NAME: SAIYED, ZAINULABEDIN MOHAMEDALI
(b) NATIONALITY: Indian
(c) ADDRESS: 304, Aamin Residency, 21-22/B Shakti Society, Danilimda
Road, Ahmedabad - 380 028, India
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it has to be performed.


Technical field of the invention:
The present invention relates to isolation of genomic DNA using magnetic nanoparticles as solid phase support. The present invention specifically relates to isolation of genomic DNA directly from the crude sample, or after preparing cell lysate and cleared cell lysate thereon. Additionally, the present invention also relates to the extraction of DNA fraction from agarose gel using magnetic nanoparticles as solid phase support.
Background and prior art:
Isolation of DNA is a prerequisite step for many molecular biology techniques. The separation of DNA from the complex mixtures in which they are often found is frequently necessary before other studies and procedure like sequencing, amplification, hybridization, detection etc.
Conventionally, extracting DNA involves cell lysis followed by removal of contaminating cellular components such as proteins, lipids and carbohydrates; and finally isolating DNA using a series of precipitation and centrifugation steps, which are difficult to automate. Improvements in methods for isolating DNA have been made and more recently, methods that rely on the use of solid phase have been proposed.
In recent years, however, magnetic particles and methods for using magnetic particles have been developed for the isolation of nucleic acid materials. Several different types of magnetic particles designed for use in nucleic acid isolation, and many of those types of particles are available from commercial sources. Such magnetic particles generally fall into either of two categories, those designed to reversibly bind nucleic acid materials directly, and those designed to do so through at least one intermediary substance.
A method is needed for isolating biological entities, particularly nucleic acids, using a magnetically responsive particle capable of rapidly and efficiently directly isolating such entities sufficiently free of contaminants to be used in molecular biology procedures.

Isolation of plasmid DNA using magnetite as a solid phase adsorbent (Analytical Biochemistry 1998; 262: 92-94) discloses a method which includes binding of plasmid DNA to the solid support (magnetite - Fe3O4) in the presence of adsorption buffer.
Application of magnetite and silica-magnetite composites to the isolation of genomic DNA (J. Chromatogr A 2000; 890: 159-166) discloses isolation of genomic DNA from maize kernels using magnetite and silica-magnetite.
US 5705628 discloses a method of separating polynucleotides, such as DNA, RNA and PNA, from a solution containing polynucleotides by reversibly and non-specifically binding the polynucleotides to a solid surface, such as a magnetic microparticle, having a functional group-coated surface.
WO 96/18731 by Deggerdal Arne et al discloses method of isolating nucleic acid from a sample by contacting said sample with a detergent and a solid support, whereby soluble nucleic acid in said sample is bound to the support, and separating said support with bound nucleic acid from the sample.
US 6368800 discloses isolation of biological target materials, particularly nucleic acids, such as DNA and RNA, from other substances in a medium using silica magnetic particles.
The present invention provides a novel technique for isolation of genomic DNA using magnetic nanoparticles as solid phase support. The present invention describes isolation of genomic DNA directly from the samples; the said sample is directly contacted with the detergent so as to lyse the cells. Since the present invention involves isolation of DNA directly from the crude sample and while the research papers reported requires the initial lysis step followed by processing of the sample and finally separating out the cell lysate by centrifugation.
The samples from which DNA isolation is optimized using the technique described in the present invention includes whole blood, buffy coat, PBMCs (Peripheral blood

mononuclear cells), cell cultures, tissue homogenate (rat brain, liver and heart), gram positive {Streptomyces flaviscleroticus), gram-negative bacteria (E. coli and S. typhii), early eukaryotes (S. cerevisiae and Dictyostelium discoideum). Additionally, extraction of DNA fragments from agarose gel has been optimized using magnetic nanoparticles as solid phase support.
The yield of genomic DNA obtained using the technique of the present invention, is either higher or equivalent to that reported earlier when silica support was used as an adsorbent. Also, the yields of isolated DNA were substantially higher in comparison with Qiagen kit.
Object of the invention:
The object of the present invention is to provide a novel technique for isolation of genomic DNA using magnetic nanoparticles as solid phase support.
Further object of the invention is to provide magnetic nanoparticles of particle size in the range of 5-100 nanometers for greater surface area on weight basis for DNA binding.
Further object of the invention is to provide a technique for isolation of genomic DNA directly from the crude sample.
It is yet another object of the invention to provide higher or equivalent yield of genomic DNA to that reported when silica support is used as adsorbent.
Summary of the invention:
The present invention relates to isolation of genomic DNA using magnetic nanoparticles as solid phase support. The mean particle size of the magnetic nanoparticles is in the range of 5-100 nanometers, thereby providing a greater surface area on weight basis for DNA binding.

In one aspect, the present invention describes a novel technique for the isolation of genomic DNA directly from the crude sample wherein the crude sample is directly contacted with detergent so as to lyse the cells. To obtain cleared cell lysate, crude sample is treated with one or more enzymes. To the cell lysate, or cleared cell lysate, magnetite, buffer and polymer are added. The mixture is further incubated and magnetic particles bound to the DNA are separated by the application of magnetic field. The magnetic pellet is washed, dried, suspended in sterile water or TE buffer and finally the supernatant containing the eluted DNA is separated from the particles by application of an external magnetic field.
In another aspect, the present invention discloses the method of extraction of DNA fragments from agarose gel. Gel electrophoresis is an important analytical technique that is also widely used for isolation and purification of specific DNA fragments. For this DNA fragments are separated on agarose gel by electrophoresis. The separated DNA is visualized on a UV transilluminator, and the band of interest is excised with a sterile blade and transferred to a microcentrifuge tube. The agarose plug containing the DNA is melted at high temperature in the presence of buffer Subsequently melted agarose plug is contacted with a solid phase support in the presence of binding buffer. The mixture is further incubated and magnetic particles bound to the DNA are separated by the application of magnetic field. The magnetic pellet is washed, dried, suspended in sterile water or TE buffer and finally the supernatant containing the eluted DNA is separated from the particles by application of an external magnetic field.
The present invention provides higher or equivalent yields of genomic DNA to that reported with conventional procedures and also when silica support is used as adsorbent. Also comparison with Qiagen kit; indicated that the yields of isolated DNA was 1.3 fold higher.
Detailed description of the invention:
The present invention describes a novel technique for the isolation of genomic DNA. The present invention relates to isolation of genomic DNA using magnetic nanoparticles as solid phase support. The mean particle size of the magnetic nanoparticles is in the range

of 5-100 nanometers, which provides a greater surface area on weight basis for DNA binding. The present invention describes a novel technique for the isolation of genomic DNA directly from the samples by contacting the sample with detergent so as to lyse the cells.
To the cell lysate magnetic particles (Fe304) are added followed by addition of binding buffer (4 M sodium chloride (NaCl) and 20 % PEG-6000). After incubating the mixture for 5 min, the magnetic particles bound to the DNA are separated from the rest of the material by application of a magnetic field. The magnetic pellet is washed twice in 50 % ethanol, followed by drying the pellet for few minutes. The pellet is finally suspended in sterile water or TE (Tris-EDTA, pH 7.8) buffer and the magnetic particles bound DNA is eluted by constant shaking and incubation at 65°C for 5 min. The supernatant containing the eluted DNA is separated from the particles by application of an external magnetic field.
The samples from which DNA isolation is optimized using this method includes, whole blood, buffy coat, PBMCs (Peripheral blood mononuclear cells), cell cultures, tissue homogenate (rat brain, liver and heart), gram positive (Streptomyces flaviscleroticus), gram-negative bacteria (E. coli and S. typhii), early eukaryotes (S, cerevisiae and Dictyostelium discoideum). Additionally, extraction of DNA fragments from agarose gel has been optimized using magnetic nanoparticles as solid phase support.
In accordance with the present invention, elution of DNA from agarose gel has also been performed using this method. The yield of genomic DNA obtained using this method was either higher or equivalent to that reported earlier when silica support was used as adsorbent.
Comparison of this method with Qiagen kit; indicated that the yields of isolated DNA was 1.3 fold higher.
The method involves isolation of DNA directly from the crude sample in contrast with the prior art where the process involves the initial lysis step followed by processing of the sample and finally separating out the cell lysate by centrifugation. The improved process

of direct isolation with higher yield is not only technologically superior but also is of economic significance. The cell lysate is further used for isolation of DNA using magnetite or silica-magnetite.
The magnetic nanoparticles used in the method of the present invention are metal oxide preferably iron oxide which displays an amphoteric hydroxy! (-OH) group on the surface. This -OH group can be used for linking variety of biomolecules through covalent coupling chemistries or under specific condition to tightly adsorb biomolecules such as DNA.
In the present invention, use of magnetic nanoparticles offers several advantages.
1. The nano-size of the particles, provide higher surface area (on a weight basis) for binding of the biomolecules.
2. The particles of the present invention are uncoated (naked), which makes them highly susceptible to the external magnetic field.
3. Due to the nano-size, the particles can exist as stable colloidal suspension that will not aggregate, allowing for uniform distribution in a reaction mixture.
The magnetic nanoparticles were prepared in the laboratory by co-precipitating di and trivalent iron (Fe) ions by alkaline solution and treating under hydrothermal conditions. The particles were stored in TE buffer (10 mM Tris -HC1 and ImM EDTA, pH 8.0) at a suspension concentration of 20 mg/ml. All agarose gels were run on 0.8% final agarose in IX TAE buffer. The field strength was 8V/cm with run times of about 60 min. The gels were stained with ethidium bromide and visualized using a UV transilluminator.
The DNA extraction method of the present invention was tested for its efficiency and ease of use compared with a commercially available kit (QlAamp DNA Blood Mini Kit for blood and QIAamp DNA Mini Kit for cultured cells, Qiagen).
The yield of DNA extracted using the method of the present invention was on average 1.3-fold greater than that using the Qiagen method. Moreover, the method of the present

invention can be carried out in a single microcentrifuge tube per sample, whereas the Qiagen procedure requires a number of tube transfers.
The higher yield of genomic DNA obtained using magnetic nanoparticles as solid-phase support may be attributed to the nano-size of the particles, which provides increased surface area for binding of DNA and creation of optimal conditions in the presence of binding buffer (4M NaCl and 20% PEG 6000).
Additionally, the DNA from all the extractions was found to function satisfactorily in restriction endonuclease digestion. This indicates absence of any enzyme inhibitors in the extracted DNA.
Furthermore, the efficiency of the extracted DNA was also checked in PCR amplification. A 226-bp fragment of GAPDH gene was successfully amplified using the genomic DNA extracted from whole blood as a template.
Following selected examples illustrates the applicability of the present invention, which are not limiting in any way.
Examples:
Example 1:
Genomic DNA isolation from whole blood using magnetic nanoparticles as a solid phase
support:
1. 30 μ,l of sample (whole blood), 30 μl of 1% (w/v) detergent (SDS) solution were added.
2. The tube was mixed by gentle inversion for two or three times and incubated at room temperature for 2 min.
3. After incubation, 10 μl of magnetic nanoparticles (20 mg/ml) were added to the cell lysate, followed by addition of 75 μl of binding buffer (4 M sodium chloride and 20% PEG 6000).
4. The suspension was mixed by inversion and allowed to stand at room temperature for at least 3 min.

5. The magnetic pellet was immobilized by application of an external magnet and supernatant was removed.
6. The magnetic pellet was washed with 50 % ethanol and dried.
7. The pellet was then completely resuspended in 50 μ1 of TE buffer (pH 8.0) by repeated pipetting strokes and magnetic particle bound DNA was eluted by incubation at 65 °C for 5 min with continuous agitation.
8. The magnetite particles were then immobilized with a magnet and the clear supernatant containing DNA was transferred to a fresh tube. The eluted DNA was analyzed using agarose gel electrophoresis.
The yields of recovered genomic DNA were up to 1.2ug per 30μl of whole blood.
The above procedure was also found to be applicable for genomic DNA isolation from buffy coat and peripheral blood mononuclear cells (PBMCs). However, in those cases only 10 (J of starting material was taken instead of 30μ.J used for blood. This is due to the fact that buffy coat and PBMCs contains only nucleated cells
Example 2:
Genomic DNA isolation from cultured cells using magnetic nanoparticles as a solid phase
support:
Cultured cells used in the present invention were of colon carcinoma cell lines (HCT116).
For genomic DNA isolation, the cells were trypsinized and adjusted to a cell density of 7
x 106 cells per ml with PBS (phosphate buffered saline, pH 7.4).
1. 30 μ1 of cell suspension, which corresponds to 2 x 105 cells were taken in a microfuge tube, followed by addition of 30 μl of detergent (1% w/v SDS).
2. Same steps as blood DNA isolation (step 2-8) were followed.
Example 3:
Genomic DNA isolation from yeast cells (S. cereviceae) using magnetic nanoparticles as
a solid phase support:
The genomic DNA from yeast cells was purified by creating a cleared lysate, reversibly
binding the genomic DNA to the magnetic nanoparticles, separating the magnetic

nanoparticles from rest of the components and finally eluting the bound DNA. The procedure used is described below:
1. S. cereviceae were grown in yeast potato dextrose (YPD) medium to a cell density of 10s cells per ml.
2. Preparation of a cleared cell lysate: Centrifuge to pellet the cells.
Supernatant was discarded and resupend the pellet in 100 ΜI sorbitol buffer
containing zymolase (200U/ml),
Incubate at 37°C for 10 min.
Treat the cell suspension with 12.5 μ.1 of detergent (10 % SDS) plus 10 u.1 of
proteinase K. (20 mg/ml) and incubate at 65°C for 10 min.
Centrifuge and transfer supernatant to a fresh tube.
3. To the cleared cell lysate, 75 μl of magnetic nanoparticles (10 mg/ml) were added followed by 200 μ.1 of binding buffer (4 M sodium chloride and 20% PEG 6000).
4. After the incubation at room temperature for 5 min, the magnetic pellet was washed twice with 50% ethanol.
5. Finally, the pellet was resuspended in 50 μl of TE buffer and DNA eluted.
Example 4:
Extraction of DNA from agarose gel using magnetic nanoparticles as a solid-phase
support:
The present invention was also found to be applicable for extraction of DNA fragments
from agarose gels:
In an optimized procedure,
1. Run DNA sample (23-kb) on a 0.8 % agarose gel.
2. Visualize the DNA band using a UV transilluminator
3. Excise the band of interest with a sterile blade and transferred to a microcentrifuge tube.
4. Add 4 volumes of SSC (0.75 M sodium chloride, 0.0075 M sodium citrate, pH 7.0) buffer to the agarose plug and incubate at 80°C for 5 min to allow agarose to melt.

5. After incubation, add 20 μl of magnetic nanoparticies (10 mg/ml), followed by addition of 200 μl of binding buffer (4M sodium chloride and 20% PEG 6000).
6. Mix the suspension by inversion and allowed it to stand at room temperature for 5 min.
7. Immobilize the magnetic pellet by application of an external magnetic field and discard the supernatant.
8. Wash the magnetic pellet twice with 50 % ethanol.
9. Resuspend the pellet in 30 μl of TE buffer and elute DNA from magnetic nanoparticle by incubation at 65 °C with continuous agitation.
10. Finally, separate the particles magnetically and transfer the supernatant containing DNA to a fresh tube.
The yield of recovered DNA from agarose gel with the method described in the present invention was >80%s whereas it was only 50 to 60% with conventional phenol extraction and glass wool spin column procedures.
Jt will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative examples and that the present invention may be embodied in other specific forms without departing from the essential attributes thereof, and it is therefore desired that the present embodiments and examples be considered in all respects as illustrative and not restrictive, reference being made to the appended claims, rather than to the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

We claim,
1. A method of isolating genomic DNA from a sample, comprising
a) contacting the DNA sample with detergent and a solid phase support,
b) separating the solid phase support bound to DNA from rest of the components by application of external magnetic field,
c) eluting the bound DNA, and finally
d) separating the supernatant containing the eluted DNA from the solid phase support by application of an external magnetic field.

2. A method as claimed in claim 1, wherein the DNA sample is selected from a group includes, but not limited to, whole blood, buffy coat, PBMCs (Peripheral blood mononuclear cells), cell cultures, tissue homogenate (rat brain, liver and heart), gram positive {Streptomyces jlaviscleroticus), gram-negative bacteria (E, coli and S. typhii), early eukaryotes (S. cerevisiae and Dictyostelium discoideum).
3. A method as claimed in claim 1, wherein the DNA sample is a crude sample.
4. A method as claimed in claim 1, wherein the DNA sample is a cell lysate.
5. A method as claimed in claim 1, wherein the DNA sample is a cleared cell lysate.
6. A method as claimed in claim 1, wherein the DNA sample is further treated with one or more enzymes to obtain cleared cell lysate.
7. A method as claimed in claim 1, wherein the detergent is selected from alkali metal alkylsulphate salt, more preferably sodium dodecyl sulphate.

8. A method as claimed in claim 1, wherein the concentration of detergent is in the range of 0.5% to 10%.
9. A method as claimed in claim 1, wherein solid phase support is uncoated particulate.
10. A method as claimed in claim 1, wherein solid phase support is a stable colloidal suspension.
11. A method as claimed in claim 1, wherein solid phase support is contained in a binding buffer.
12. A method as claimed in claim II, wherein binding buffer comprises a mixture of one or more monovalent salts such as sodium chloride or sodium bromide or a mixture thereof, in the concentration of about 1M to 5M and the polymer such as polyethylene glycol, preferably in the concentration of about 15% to 25%.
13. A method as claimed in claim 12, wherein the polythene glycol has a
molecular weight between 4000 to 10,000, preferably 6000.
14. A method as claimed in claim 12, wherein the proteins are precipitated from the solution by altering the dielectric constant of the solution by addition of polyethylene glycol
15. A method as claimed in claim 1, wherein solid phase support comprises magnetic nanoparticles.
16. A method as claimed in claim 1, wherein solid phase support further has an amphoteric hydroxyl (-OH) group on the surface.

17. A method as claimed in claim 15, wherein magnetic nanoparticles are metal oxide, preferably divalent and trivalent iron oxides.
18. A method as claimed in claim 15, wherein the particle size of magnetic nanoparticles range from 5 to 100 nanometers.
19. A method as claimed in claim 1 , wherein the genomic DNA bound to solid phase support forms magnetic pellets at room temperature.
20. A method as claimed in claim 1 , wherein magnetic pellets are immobilized using external magnetic field.
21. A method as claimed in claim 1 , wherein magnetic pellets having DNA bound thereto are contacted with elution buffer.
22. A method as claimed in claim 21 , wherein elution buffer is Tris hydrochloride and EDTA .
23. A method as claimed in claim 21 , wherein the pH of the elution buffer ranges from 7.5 to 8.5 pH.
24. A method as claimed in claim 1 , wherein the genomic DNA is eluted by heating at the temperature ranging from 50°C to 70°C, preferably 65°C.
25. A method of extraction of DNA fragments from agarose gel, comprising

a) separating DNA fragments by agarose gel electrophoresis
b) melting the excised agarose plug containing DNA fragments in presence of buffer,
c) contacting melted agarose plug with a solid phase support and binding buffer,
d) separating the solid phase support bound to DNA from rest of the
components by application of external magnetic field,

e) eluting the bound DNA, and finally
f) separating the supernatant containing the eluted DNA from the solid phase support by application of an external magnetic field.
Dated this 4th day of July, 2007


Documents:

1067-mum-2006-abstract(4-7-2007).doc

1067-mum-2006-abstract(4-7-2007).pdf

1067-mum-2006-abstract(granted)-(25-11-2010).doc

1067-mum-2006-abstract(granted)-(25-11-2010).pdf

1067-mum-2006-cancelled pages(25-10-2010).pdf

1067-mum-2006-claims(4-7-2007).doc

1067-mum-2006-claims(4-7-2007).pdf

1067-MUM-2006-CLAIMS(AMENDED)-(25-10-2010).pdf

1067-MUM-2006-CLAIMS(AMENDED)-(29-7-2010).pdf

1067-mum-2006-claims(granted)-(25-11-2010).doc

1067-mum-2006-claims(granted)-(25-11-2010).pdf

1067-MUM-2006-CLAIMS(MARKED COPY)-(25-10-2010).pdf

1067-MUM-2006-CLAIMS(MARKED COPY)-(29-7-2010).pdf

1067-MUM-2006-CORRESPONDENCE(25-10-2010).pdf

1067-MUM-2006-CORRESPONDENCE(28-7-2008).pdf

1067-mum-2006-correspondence(4-7-2007).pdf

1067-mum-2006-correspondence(ipo)-(11-12-2009).pdf

1067-mum-2006-correspondence(ipo)-(25-11-2010).pdf

1067-mum-2006-correspondence-others.pdf

1067-mum-2006-description(complete)-(4-7-2007).pdf

1067-mum-2006-description(granted)-(25-11-2010).pdf

1067-mum-2006-description(provisional).pdf

1067-mum-2006-form 1(15-1-2007).pdf

1067-MUM-2006-FORM 1(25-10-2010).pdf

1067-MUM-2006-FORM 1(29-7-2010).pdf

1067-mum-2006-form 1(6-7-2006).pdf

1067-MUM-2006-FORM 18(28-7-2008).pdf

1067-mum-2006-form 2(4-7-2007).doc

1067-mum-2006-form 2(4-7-2007).pdf

1067-mum-2006-form 2(granted)-(25-11-2010).doc

1067-mum-2006-form 2(granted)-(25-11-2010).pdf

1067-MUM-2006-FORM 2(TITLE PAGE)-(25-10-2010).pdf

1067-MUM-2006-FORM 2(TITLE PAGE)-(29-7-2010).pdf

1067-mum-2006-form 2(title page)-(4-7-2007).pdf

1067-mum-2006-form 2(title page)-(complete)-(4-7-2007).pdf

1067-mum-2006-form 2(title page)-(granted)-(25-11-2010).pdf

1067-mum-2006-form 2(title page)-(provisional)-(6-7-2006).pdf

1067-mum-2006-form 2.doc

1067-mum-2006-form 26(12-1-2007).pdf

1067-MUM-2006-FORM 3(25-10-2010).pdf

1067-MUM-2006-FORM 3(29-7-2010).pdf

1067-mum-2006-form 3(6-7-2006).pdf

1067-mum-2006-form 5(4-7-2007).pdf

1067-mum-2006-form-1.pdf

1067-mum-2006-form-2.pdf

1067-mum-2006-form-3.pdf

1067-MUM-2006-PETITION UNDER RULE 137(25-10-2010).pdf

1067-MUM-2006-PETITION UNDER RULE 137(29-7-2010).pdf

1067-MUM-2006-REPLY TO EXAMINATION REPORT(25-10-2010).pdf

1067-MUM-2006-REPLY TO EXAMINATION REPORT(29-7-2010).pdf

1067-mum-2006-specification(amended)-(29-7-2010).pdf


Patent Number 244248
Indian Patent Application Number 1067/MUM/2006
PG Journal Number 49/2010
Publication Date 03-Dec-2010
Grant Date 25-Nov-2010
Date of Filing 06-Jul-2006
Name of Patentee SAIYED, ZAINULABEDIN MOHAMEDALI
Applicant Address 304, AAMIN RESIDENCY, 21-22/B SHAKTI SOCIETY, DANILIMDA ROAD, AHMEDABAD-380 028,
Inventors:
# Inventor's Name Inventor's Address
1 SAIYED, ZAINULABEDIN MOHAMEDALI 304, AAMIN RESIDENCY,21-22/B SHAKTI SOCIETY, DANILIMDA ROAD, AHMEDABAD-380 028,
2 CHANIYILPARAMPU, NANAPPAN RAMCHAND K 104,16th Street, Annanagar East, Chennai-600 102,India
3 KAPOOR, AJAY 73,Hallamshire Road, Sheffield S10 4FN,
PCT International Classification Number C07H19/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA