|Title of Invention||
"A NON-INVASIVE DNA ISOLATION METHODOLOGY IN DAIRY ANIMALS"
|Abstract||This invention relates to a novel process for purifying and/or isolating nucleic acids from fecal/epithelial tissue in cattle and buffalo comprising disrupting the dead fecal epithelial tissue, selectively protecting the nucleic acids and finally purifying them by: i) washing of faeces with mucous membrane with sterile phosphate buffered saline, removing excess water carefully by micropipette ii) grinding in presence of liquid nitrogen, extracting with SOmMtris pH 8.0, 30 mM EDTA and 450 mM Nacl, Homogenizing for 5 minutes on a vortex mixture. iii) Adding 120 µl 10% SDS and 15 ul proteinasek 10 mg/ml and incubating at 60°C for 3 hrs and precipitating proteins and cellular debris by 400 µl SMNacl and incubating in ice for 15 mins. iv) Centrifuging at room temperature at 12,000 rpm for 20 mins, adding supernatant to a tube containing 250 µl of gnanidine hydrpchloride 8M, pH80, 250 |al ammonium acetate (0.50 m) and 10 µl RNAse A (Img/ml), mixing gently at room temperature (20°C) for 90 mins. v) Precipitating nucleic acid by adding 0.5 volumes of chilled isopropyl alcohol and centrifugation at 7000 rpm for 5 mins at 4°C.|
|Full Text||Field of the Invention;
The invention relates to a novel process for purifying and/or isolating nucleic acids from fecal/epithelial tissue in cattle and buffalo.
Background of the Invention;
Genomic DNA acts as a major starting material for any kind of molecular genetic work like, identification, paternity control, genetic testing, QTL analysis, etc. Common genomic DNA isolation is based on a procedure in which the source material is either blood or any other soft tissue. In diary animals, different types of source materials are used like, the traditional blood sample, ear tag, hair follicles, semen, milk (Lipkin et al., 1993) etc. For forensic purposes, DNA is isolated from traces of tissue samples present in blood stains, fingerprints, dental remains, skeletal evidence, specimens from fires, explosions, airplane crashes, and other traumatic events. For most mammalian species, DNA isolation from blood is a universal and widely used method for any type of molecular biology work, as this method gives a good quality and quantity of DNA and a little amount of blood is enough to get the required amount of DNA. Blood is easily available and can be collected without harming the animal. Mammalian tissue exhibit a considerable variation in their gross and microscopic structures; hence a single DNA extraction technique is not suitable for all tissue types. Many techniques are current used for genomic DNA isolation depending upon the type, nature and amount of tissue sample available (Blin and Stafford), 1976; Bowtell, 1987; Gustafson et al., 1987; Kupiec et al., 1987; Sambrook et al., 1990; Roe et al., 1996; Singh et al., 2005). DNA extraction methods from such a
variety of of biological samples involves three basic steps. The first step is the lysis of the animal cell, nuclear membrane and mitochondrial membrane to release genomic DNA mitochondrial DNA and cytoplasmic RNA. This is achieved by a strong anionic detergent such as sodium dodecyl sulfate (SDS) by the disruption of cell membranes. Since DNA is very robust, it is not adversely affected by SDS treatment. The lysis step is assisted by Proteinase K, a serine protease which digests the protein and frees the DNA from any proteins associated with it (euchromatin structures/histones, etc.). The second step is the removal of (separation of) complex macromolecules (protein, lipid and polysaccharides) from aqueous solutions containing nucleic acids, upon treatment with organic solvents such as phenol/guanidine hydrochloride, followed by centrifugation. Buffer saturated phenol at a neutral pH brings all macromolecules together at the buffer-phenol interface. Finally, the DNA
is precipitated out of the aqueous solution by alcohols in the presence of mild cationic salts. Some commercial methods/kits are available which avoid or reduce the last two steps and the DNA is allowed to bind to a solid matrix or silica by favoring hydrophobic or ionic interactions. The bound DNA is selectively eluted after extensive washing to remove the impurities.
The development of non-invasive genetic sampling is increasingly popular/important to field biologists as the methods offer new ways to obtain genomic DNA for molecular analysis. Non-invasive sources of DNA include hair (in primates, bears, and dairy animals), milk, semen, urine and faces. Hair and feces are the most common non invasive sources of mammalian DNA. The epithelial cells shed from the intestinal lining and deposited on the surface of the feces could be considered another important non invasive source of mammalian DNA, which is retrievable
from exfoliated intestinal epithelial cells in feces. Fecal DNA isolation was reported from a variety of animals such as primates (Utami etal., 2002), lions (Ernest et al., 2000), coyotes (Kohn et ah, 1999), bears (Tablerlet et al 1997), ungulates (Flagstad et. al, 1999), dolphins, bats (Veg. and Mccracken 2001 and rhinos with varying success rates. Fecal samples are the only resource for genetic and ecological studies of wild and captive animals (Constable et ah, 1995; H'oss et ah, 1992; Kohn and Wayne, 1997, Zhang et al., 2006). The quality and quantity of the available DNA depends on the type of sample, their collection methods, preservation conditions and procedures applied to isolate the DNA. Commercial kits are available (QIAamp DNA stool Mini Kit, Qiagen) for DNA extraction. However, these kits are expensive and sometimes reported to be ineffective in herbivores (Zhang et ah, 2006). There are no reports available for DNA isolation methods from fecal tissue in dairy animals.
Normal DNA isolation procedure from farm animals:
Presently, genomic DNA is isolated by invasive methods like blood (venipuncture), ear tag, semen (in case of bull), milk (rare, in case of milking female). The remaining DNA isolation methodology is standard. We used a novel approach/protocol to process the fecal mucus tissue/dead tissue to obtain the genomic DNA.
Use of Fecal materials for the isolation of DNA and PCR:
The use of fecal material from humans as a means to obtain genomic DNA for use in genetic analysis, identification of infectious microorganisms and the diagnosis of cancerous and pre-cancerous
colorectal lesions has been reported by various groups. Non-invasive sources of DNA include hair (in primates, bears, and dairy animals), milk, semen, urine and faces. Hair and feces are the most common non invasive sources of mammalian DNA. The epithelial cells shed from the intestinal lining and deposited on the surface of the feces could be considered another important non invasive source of mammalian DNA, which is retrievable from exfoliated intestinal epithelial cells in feces. Fecal DNA isolation was reported from a variety of animals such as primates (Utami etal., 2002), lions (Ernest et al., 2000), coyotes (Kohn et al., 1999), bears (Taberlet et al 1997), ungulates (Flagstad et. al, 1999), dolphins, bats (Veg and Mccracken 2001 and rhinos with varying success rates. Fecal samples are the only resources for genetic and ecological studies of wild and captive animals (Constable et al., 1995; H"oss et al., 1992; Kohn and Wayne, 1997, Zhang et al., 2006) The quality and quantity of the available DNA depends on the type of sample, their collection methods, preservation conditions and procedures applied to isolate the DNA. Commercial kits are available (QIAamp DNA Stool Mini Kit, Qiagen) for DNA extraction. However, these kits are expensive and sometimes reported to be ineffective in herbivores (Zhang et al., 2006). There are no reports available for DNA isolation methods from fecal tissue in diary animals.
Drawbacks in invasive procedures:
The existing approaches require invasive means of collecting tissue for genomic DNA isolation. For the collection of sample for genomic DNA isolation from ear tags/peripheral blood, the animal has to be restrained in a special crate. Moreover, manpower is required to handle the animal
while collecting the tissue sample invasively. It is a big problem to collect sample by invasive means in aggressive animals and bulls.
Drawbacks in isolating DNA from feces;
The presence of inhibitors in fecal materials is a major obstacle limiting the usefulness of the PCR for detecting microorganisms in feces. A number of inhibitors are present in human feces which include bile salts, hemoglobin degradation products, and complex polysaccharides (Monteiro, L., D. Bonnemaison, A. Vekris, K.G. Retry, J. Bonnett, R. Vidal, J. Cabrita, and F. Megraud. 1997. Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model. J. Clin. Microbiol. 35:995-998.). in addition, polyphenolic substances from plant tissues are inhibitory to PCR (Koonjul, P.K., W.F. Brandt, J.M. Farrant, and G.G. Lindsey. 1999. Inclusion of polyvinylpyrrolidone in the polymerase chain reaction reverses the inhibitory effects of polyphenolic contamination of RNA. Nucleic Acids Res. 27:915-916).
It is a completely noninvasive method with enough sources for DNA;
This method does not require invasive tissues like blood and ear tags used routinely for DNA isolation (in cattle and buffalo). At present no alternative noninvasive method exists for genomic DNA isolation in Dairy animals. The amount of tissue available from the surface of the any fecal matter is a sufficient source for DNA.
Objects of the Invention:
The object of this invention is to develop a simplified DNA isolation method in cattle and buffalo.
Other object is to use a non-invasive DNA isolation methodology in Dairy animals.
Another object is to use Genomic DNA from fecal/epithelial tissue in cattle and buffalo.
Other object is to isolate nucleic acid which are found to be considerably intact and not sheared.
Another object is to avoid use of strong organic reagents such as phenol which is a inhibitor of PCR.
Yet another object is to isolate DNA from sufficient amount of available tissue.
Other object is to use fecal epithelial dead tissue for DNA isolation wherein it is resistant to any common enzymatic treatment because of its outer protective mucus layer.
Another object is to remove any probable inhibitors of DNA modifying enzymes for further processing of the genomic DNA.
Yet another object is to develop a process that can be used for DNA isolation from any tissue.
Brief description of accompanying Figs;
Fig 1. Shows gel electrophoresis of genomic DNA isolated from cattle fecal epithelial tissue.
Fig 2. Shows gel electrophoresis of genomic DNA isolated from buffalo fecal tissue.
Fig 3. Shows Restriction Enzymatic digestion of isolated genomic DNA of cattle.
Fig 4. Shows PCR amplification of mitochondrial DNA from isolated buffalo and cattle.
Detailed description of the invention;
The invention relates to a process for purifying and/or isolating nucleic acids from samples of domestic animal feces (cattle and buffalo), which may contain impurities and inhibitors or unwanted substances. The invention describes a detailed procedure and reagents for carrying out the said method. The basis of the invention is a method of disrupting and dead fecal epithelial tissue, while selectively protecting the nucleic acids and finally purifying them. The isolated nucleic acids were found to be considerably intact (not broken/sheared while in the process of isolation). The isolated DNA was processed further for use in PCR and restriction endonuclease digestion methodologies.
The methodology; Sample collection;
Fecal epithelial cells were collected from freshly defecated material from the experimental animal herd of National Dairy Research Institute (Karnal, Haryana, India). The shiny surface layer on the surface of, and between, the folds of the fecal material invariably contains enough exfoliated epithelial tissue from the intestinal lumen. About 10-50 g of faeces with mucous membrane was obtained in an air tight container and transported to the laboratory for processing. The tissue was thoroughly/repeatedly washed in sterile phosphate buffered saline to remove the attached fecal material. Before further processing of the fecal tissue, the excess water was removed carefully by a micropipette. Approximately, 500 mg of tissue was processed each time. For comparative study, we obtained the skin, muscle, liver and brain tissue from an abattoir. An equal amount of skin, muscle, liver and brain tissue was also processed simultaneously for comparative study.
Approximately 200 mg of faecal epithelial tissue (or 200 mg tissued from other organs) were ground with a mortar and pestle in the presence of liquid nitrogen. The frozen powder was transferred to a 2 mL microfuge tube containing 900 µl of the extraction solution (50 mM Tris, pHS.O; 30 mM EDTA and 450 mM NaCI). The content was homogenized for 5 minutes on a vortex mixture. Subsequently, 120 µl of 10% SDS, and 15 µl Proteinase K (10 mg/ml) were added to the tissue homogenate and incubated at 60°C for 3 h. Proteins and cellular debris were precipitated
by adding 400 µl 5 M NaCI, and incubating the mixture in ice for 15 min. Centrifugation was done in room temperature at 12,000 rpm for 20 min to separate protein. About 500 µl of undisturbed supernatant were transferred to a fresh microfuge tube containing 250 µl of guanidine hydrochloride (8 M, pH 8.0), 250µl of ammonium acetate (0.50 M) and 10 µl RNAse A (Img/ml). The content was mixed gently and incubated at room temperature (20°C) for 90 min. Nucleic acid was precipitated by adding 0.5 volumes of chilled Isopropyl alcohol and centrifugation at 7000 rpm for 5 min at 4°C temperature. The pellet was washed with 500 µ1 freshly prepared 70% Ethyl alcohol and dissolved in 100 pi TE buffer (10 mM Tris and ImM EDTA, pH 8.0). DNA samples were stored at 4°C for further use. DNA quality and concentration were evaluated by reading their absorbance at 260 nm in a UV-VIS spectrophotometer (Nano drop) and agarose gel electrophoresis (1%).
Results show that high molecular weight DNA could be isolated by this process. This new protocol was tested by comparison with a DNA isolation method successfully used for several animal tissues in our laboratory (Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.). Using a normal/routine DNA isolation procedure, Inventors were unable to obtain DNA from this fecal tissue. The experiment was repeated many times to get traces of DNA, but we could not succeed. This could be due to improper lysis of faecal tissue/cells. Subsequently we started modifying the process and standardized this protocol. While collecting the tissues, plant materials were removed by several washings with PBS which eliminated the use of PVP (polyvinylpyrrolidone) and b-mercaptoethanol (which is normally recommended for DNA isolation from faecal tissue containing undigested
plant residues) which prevent oxidation of the secondary metabolites present in the disrupted plant material. Moreover, this procedure could be extended successfully to several other types of tissue tested (like liver, kidney, brain, skin etc) (Figure 2). However, no significant differences in the quality and yield of isolated DNA could be detected in other tissue.
Quality and quantity of DNA;
In agarose gel electrophoresis the isolated DNA revealed a single band indicating a considerably uniform high molecular weight (Figure I). The DNA extracted with this protocol showed digestability with restriction enzymes. The digested DNA revealed a characteristic 'smear' (Figure 3). Thus; DNA isolated with this procedure can be used for most molecular procedures. The isolated DNA was tested for PCR amplification of mitochondrial DNA segments (Figure 4) without problem. Therefore, this DNA can also be used in molecular ecology analyses (e.g. population genetics). The quantity of the isolated DNA was estimated by the UV-VIS spectrophotometer (Nanodrop). The isolated DNA had normal spectra in which the A260/A280 ratios were 1.6-1.7 (Table 1).
Table 1- DNA concentration (A260nm and OD ratio (A2eonm/A280nm) of DNA extracted from different tissues.
The present work describes the suitability of a new methodology to isolate genomic DNA from exfoliated intestinal cells available in the feces of cattle and buffalo. This protocol is also found suitable for a variety of tissue samples, like muscle, liver, kidney and blood. Speed and throughput of the technique enable large numbers of samples to be processed quickly without the use of strong organic solvents such as phenol.
It is to be noted that the formulation of the present invention is susceptible to modifications, adaptations and changes by those skilled in the art. Such variant formulations are intended to be within the scope of the present invention which is further set forth under the following claims:-
1. A novel process for purifying and/or isolating nucleic acids from fecal/epithelial tissue in cattle and buffalo comprising disrupting the dead fecal epithelial tissue, selectively protecting the nucleic acids and finally purifying them by:
i) washing of faeces with mucous membrane with sterile phosphate buffered saline, removing excess water carefully by micropipette
ii) grinding in presence of liquid nitrogen, extracting with SOmMtris pH 8.0, 30 mM EDTA and 450 mM Nacl, Homogenizing for 5 minutes on a vortex mixture.
iii) Adding 120 μl 10% SDS and 15 jil proteinasek 10 mg/ml and incubating at 60°C for 3 hrs and precipitating proteins and cellular debris by 400 jal SMNacl and incubating in ice for 15 mins.
iv) Centrifuging at room temperature at 12,000 rprn for 20 mins, adding supernatant to a tube containing 250 μl of gnanidine hydrochloride 8M, pH80, 250 jal ammonium acetate (0.50 m) and 10 al RNAse A (1 mg/ml), mixing gently at room temperature (20°C) for 90 mins.
v) Precipitating nucleic acid by adding 0.5 volumes of chilled isopropyl alcohol and centrifugation at 7000 rpm for 5 mins at 4°C.
2. The process of DNA isolation as claimed in claim 1, wherein
slimy tissue is collected from the surface and the folds of cake
type feces, which retains much water and normally not
susceptible to the cell lytic chemicals.
3. The process of DNA purification as claimed in claim 1, wherein
the final DNA pellet is washed with 500 jul freshly prepared 70%
4. The process of DNA isolation as claimed in claim 1, wherein
fecal epithelial cells are collected from freshly defecated
|Indian Patent Application Number||1366/DEL/2007|
|PG Journal Number||05/2014|
|Date of Filing||26-Jun-2007|
|Name of Patentee||NATIONAL RESEARCH DEVELOPMENT CORPORATION|
|Applicant Address||“ANUSANDHAN VIKAS" 20-22, ZAMROODPUR COMMUNITY CENTRE, KAILASH COLONY EXTENSION, NEW DELHI-110 048|
|PCT International Classification Number||C12Q1/68|
|PCT International Application Number||N/A|
|PCT International Filing date|