WO2000073508A1 - Methods and compositions for isolating high molecular weight dna from natural samples - Google Patents
Methods and compositions for isolating high molecular weight dna from natural samples Download PDFInfo
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Definitions
- the present invention relates to novel methods for the isolation and cloning of high molecular weight DNA collected from a variety of natural sources and a DNA library produced therefrom. More particularly, the invention relates to the isolation of DNA from a plurality of species collected from natural sources to produce a library of high molecular weight DNA fragments generated from those organisms, either singly or in recombination with DNA fragments from other species.
- Natural products possessing other biological activities of human benefit have been discovered as well. These include anticoccidial agents, anti-fungal drugs, herbicidal agents, anticancer drugs, insecticidal and nematocidal agents, immunomodulating compounds, and enzyme inhibitors.
- microorganisms produce a variety of lipopeptides, lipoproteins, glycolipids, and lipopolysaccharides with surface-acting properties (Rosenberg, 1986).
- cellulases, amylases, proteases, and lipases used extensively in textile applications.
- Other microbial enzymes are important in the biotechnology industry (i.e., restriction enzymes and thermostable enzymes).
- Recovery of genomic DNA from a natural sample may be accomplished by the isolation of organisms from a natural sample, culturing the organisms, and extracting gDNA from the culture.
- This method has a number of disadvantages, however. The process is time consuming and requires a large amount of practitioner manipulation and person hours. In addition, the process requires prior knowledge of the organism's physical, chemical, and biological requirements for successful culturing. Alternatively, the practitioner may select arbitrary culture conditions in an effort to culture whatever may grow under the selected conditions. As mentioned earlier, this is a significant limitation, since it is believed that the vast majority of the Earth's biota remains uncharacterized and unknown.
- Recovery of genomic DNA from a natural sample may also be accomplished by DNA isolation directly from the collected natural sample.
- the organisms of the sample are not cultured or otherwise isolated from each other or their natural environment.
- This direct DNA extraction method lacks robustness in regard to the quality and quantity of the extracted gDNA, however:
- the quantity of gDNA isolated by conventional direct extraction methods is small, requiring either large amounts of initial sample or repeat sampling. Obtaining large amounts of a natural sample may be impossible or impractical, especially if the sample is taken from remote locales or environmentally sensitive habitats.
- laboratory manipulation of large amounts of sample during the DNA extraction protocol is impractical. Repeat sampling suffers from these same limitations.
- repeat sampling does not guarantee successful repeated isolation of the same DNA fragments.
- gDNA isolated by direct extraction methods is also limited. Despite years of scientific effort, the extraction of high molecular weight gDNA (i.e., >50 kilobase pairs) directly from natural samples has not been demonstrated. The isolation of large gDNA fragments is necessary, however, for proper forensic or phylogenetic analysis, as well as for the discovery of larger polypeptides or compounds produced from the biological activity of a plurality of polypeptides (e.g., a biochemical pathway).
- a method is needed that would allow DNA extraction directly from a variety of natural samples, without the need to culture the organisms of the sample prior to DNA extraction.
- this method would allow direct DNA extraction without requiring large amounts of initial sample, or repeat sampling.
- this improved method would be able to isolate high molecular weight DNA from a sample, and in a form and condition whereby the high molecular weight DNA can be retained, sequenced, and expressed for further research and development.
- the present invention is directed to a novel method for recovering high molecular weight DNA (hmwDNA) from a natural sample.
- hmwDNA high molecular weight DNA
- the sample will contain a plurality of species.
- the present invention offers several advantages and novel features over DNA extraction techniques known in the art.
- the present invention provides the advantage of improved sensitivity, thus not requiring a large amount of initial sample collected from the natural environment.
- the invention employs equipment and reagents known and used by persons of ordinary skill in the art, and does not require the use of expensive, cumbersome, or otherwise exotic equipment or reagents.
- the method of the present invention requires less sample manipulation than current techniques known in the art.
- the method allows for DNA extraction directly from a natural sample, without the need to culture organisms contained within the sample, or any other pre-treatment of the sample before the process of DNA extraction. This invention, therefore, reduces the time and expense of additional reagents, equipment, incubation time, and practitioner manipulation.
- the method of the present invention provides a more precise representation of the total genetic diversity contained within a given sample than conventional DNA isolation techniques known in the art. Because DNA extraction may commence immediately after sample acquisition, there is less opportunity for the degradation of some genetic material (e.g., due to organism die-off, DNA hydrolysis, etc.) and the amplification of other genetic material (e.g., organism proliferation).
- the improved techniques of the present invention require no prior understanding or knowledge of the biological requirements (or even the existence) of the organisms in the sample to be processed. (See, for example, Torsvik et al., 1990 and 1994.)
- the present invention provides for the extraction of hmwDNA from a natural sample.
- DNA isolated by the present invention can range in size from 50,000 to 400,000 base pairs (50-400 kbp). As a result, the present invention can produce single DNA fragments equivalent in length to one tenth of a typical bacterial genome. Extraction of DNA fragments of this magnitude from a natural sample is not known in the art. The isolation of high quality DNA is critical for phylogenetic or forensic analyses. hmwDNA is essential for the discovery of larger polypeptides, or polypeptides encoded by gDNA that contain noncoding regions (e.g., introns).
- hmwDNA The isolation of hmwDNA is also necessary for the discovery of polypeptides formed from two or more heterogeneous polypeptide subunits, or other gene clusters and their products; for example compounds produced as a result of a biochemical pathway requiring two or more polypeptides
- the method of the present invention provides for the isolation from a natural sample of hmwDNA fragments suitable for incorporation into a genetic vector, transgenic incorporation into a host organism, and subsequent expression of the DNA insert. It is therefore another object of the invention to provide a library of hmwDNA isolated from a natural sample.
- the library of the present invention provides for the unlimited storage of the DNA inserts and the genetic information contained therein, and eliminates the need or necessity to obtain additional samples from the natural environment.
- the library of the present invention can be utilized in the analysis and screening of the genetic information, or the expressed polypeptides for a wide variety of research and development applications (e.g., phylogenetic analyses and drug discovery programs as described earlier).
- the method of the present invention essentially comprises: preparing an aqueous suspension of a natural sample, gently emulsifying the suspension with an organic solvent, and precipitating the DNA for solution.
- the extraction process comprises additional steps, before the final DNA precipitation, including washing the sample solution with a cationic detergent, and additional organic solvent separations.
- the extraction process culminates with a gradient separation step, to separate the isolated DNA on the basis of molecular weight. It is critical to the invention that the steps of the process are carried out gently, to minimize or prevent shearing of the sample DNA.
- This invention provides a method for isolating high molecular weight DNA from a plurality of species in a sample, including environmental samples such as soil samples or other samples of material from nature.
- the method involves suspending a portion of the sample in an aqueous medium to preparing an aqueous suspension of the sample. Then, an extraction mixture is formed by adding to the aqueous suspension an appropriate organic solvent under suitable conditions to remove undesired materials from the suspension while retaining part or all of the high molecular weight DNA.
- Suitable solvents include phenol, or other organic solvents capable of dissolving undesired materials such as proteins, lipids, etc.
- the phenol or other organic solvent is sufficiently warm, usually >50° C in the case of phenol, and is added under suitable conditions (preferably gentle mixing) to dissolve the undesired materials.
- gentle we mean conditions sufficiently vigorous for dissolution and removal of the undesired materials from the aqueous phase which contains the high molecular weight DNA, yet gentle enough so that at least part, and preferably most, of the high molecular weight DNA remains as such in the aqueous phase.
- the extraction mixture is then separated into an aqueous phase and an organic phase.
- the aqueous phase contains the high molecular weight DNA, which may then be precipitated from the aqueous solution using conventional methods and materials, e.g., addition of a cosolvent such as an alcohol, generally ethanol or isopropanol.
- a cationic detergent may be added to the separated aqueous phase to form an aqueous mixture.
- a preferred cationic detergent for this use is cetyltrimethylammonium bromide (CTAB).
- CTAB cetyltrimethylammonium bromide
- the aqueous mixture may then be gently extracted with an organic solvent, which may be the same or different from the solvent used in the previous extraction step. Often the organic solvent used to remove the detergent is chloroform, but phenol or any other suitable solvent may be used.
- the invention optionally further comprises passing the extracted DNA over a density gradient to separate the hmwDNA from smaller DNA fragments.
- the solution containing the DNA Prior to putting the DNA on the gradient, the solution containing the DNA must be concentrated, either by reducing the volume of the solution or by precipitating the DNA from solution and resuspending it in a smaller volume. If desired, the practitioner may further remove contaminants from the hmwDNA by adding a proteinase to the DNA in a sufficient amount and under appropriate conditions permitting degradation of proteins.
- the present invention also provides for a genetic construct comprising hmwDNA isolated from a natural sample incorporated into a cloning vector.
- the DNA insert-vector construct is stable and capable of replication of the DNA insert.
- the genetic construct is capable of expressing a polypeptide encoded by the hmwDNA insert.
- the present invention further provides for a transgenic host cell comprising the incorporation of hmwDNA isolated from a natural sample into a living host cell.
- the hmwDNA is stably incorporated into a cloning vector.
- the hmwDNA is stably incorporated into a host cell chromosome.
- the host cell is capable of expressing one or more polypeptide(s) encoded by the hmwDNA insert.
- Fig. 1 is a photograph of an ethidium bromide (EtdBr)-stained agarose gel, depicting DNA isolated from a natural soil sample.
- Lane 1 is a nucleic acid ladder, used as a gel reference marker.
- Lane 2 shows total genomic DNA extracted from a natural soil sample prior to density gradient separation.
- Lane 3 represents a soil hmwDNA fraction separated by sucrose gradient centrifugation.
- Lanes 4 and 5 show restriction digests of soil hmwDNA (prior to vector ligation). Lane is soil hmwDNA cut with EcoRl, and lane 5 is soil hmwDNA cut with Hindlll.
- Fig. 2 is a diagram of pBTP2 used for the construction of the soil library.
- Vector pBTP2 is a modification of pBeloBACl 1, containing additional cloning sites and a pUC origin of replication inserted into the polylinker (allowing for high-copy replication of the empty vector to facilitate purification). The pUC sequence is removed by two sequential gel purification steps before ligation with insert DNA.
- B Cloning site of pBTP2. Uppercase letter indicate pBeloBac sequences, lowercase letters indicate PUC 19 sequences, and Bold sequences identify the pBacTA polylinker.
- Fig. 3 is a phylogenetic tree construct based on small subunit (16S) rRNA sequence homology of DNA obtained from soil, and constructed using a Phylogenetic Inference algorithm (PHYLIPTM, version 3.57, J. Felsenstein, U. of Washington, Seattle). Incorporated into the diagram are known bacterial representatives of divergent taxonomic families, including several from published reports of 16S subunit sequence analysis of DNA obtained directly from soil.
- PHYLIPTM Phylogenetic Inference algorithm
- Fig. 4 is an ORF map of clone MG1.1. Sequence was determined as described in the methods. ORF's were identified using MapDraw (DNAStar Inc.). Homology search was done using BLAST
- Fig. 5 illustrates the one-dimensional proton NMR spectrum of red compound (A) compared to a standard sample of Indirubin (B) (Sigma). Both spectra are recorded in ⁇ VDMSO solvent at 27°C).
- Panel C illustrates the structure, determined by NMR, of a colorless compound isolated from MG1.1 with anti-bacterial activity (2-(2,2-bis-(lH- indol-3-yl)-ethyl)-phenylamine).
- the present invention provides a novel method for recovering hmwDNA from a natural sample.
- DNA is a polymer of nucleotide bases
- DNA molecular weight is directly proportional to polymer size. Therefore, as used herein, the term high molecular weight DNA (hmwDNA) refers to DNA comprising a polymer of nucleotides at least about 50,000 base pairs in length (> 50 kbp).
- hmwDNA ranges between about 50 kbp to about 400 kbp. More preferably the DNA length is about 80 kbp to about 300 kbp.
- a natural sample refers to any sample taken from the natural environment.
- the natural environment is meant to encompass the biosphere, or any environment wherein genetic material from an organism may be found.
- the natural sample will contain genetic material from a plurality of species.
- Natural samples include but are not limited to samples taken from any soil (encompassing all of the soil types and depths), water (encompassing all freshwater aquatic, or marine habitats), or atmospheric environment. Sampling techniques are well known in the art (see for example Colwell, 1979; Fenical and Jenson, 1992; Giovanni et al., 1990; Griffiths et al., 1996; Stahl et al., 1985; Suzuki et al., 1997; Torsvik et al. 1994; Ward, 1990).
- a natural sample may also comprise a sample (apparently) taken from a single organism, (such as plant or animal samples that may contain genetic material from more than one species due to infestation or symbiosis; see for example Currie et al., 1999).
- the genetic diversity contained within a natural sample will depend upon the species diversity of the sample and may vary depending upon when the sample is taken as well as where it is taken. Circannual, seasonal, and even circadian changes in the biodiversity of a natural habitat will affect the diversity of genetic material of the present invention isolated from a sample.
- a natural sample contains a multitude of species, thus increasing the total genetic diversity contained within the sample. Most preferably, the sample contains a multitude of previously uncharacterized and unknown species.
- species refers to any taxonomic grouping of genetically distinct individuals. Independent of any ongoing taxonomic debate, viruses are included in the definition of species, as used herein. Species, and individuals of a species, need not be living organisms, but merely possess genetic material in the form of nucleic acid.
- a DNA library refers to a compilation of genetic constructs, each comprising a DNA fragment stably inserted into a genetic vector.
- the DNA insert-vector construct is capable of replication within a host organism.
- a DNA expression library refers to a DNA library, wherein the DNA fragment is operably inserted into an expression vector, such that the DNA fragment is capable of being transcribed and translated into a polypeptide.
- gDNA genomic DNA
- hmwDNA hmwDNA
- DNA extraction procedures typically involve cell lysis and digestion with a combination of a proteolytic enzymes and non-ionic or anionic detergents, such as SDS.
- the DNA is isolated from the digest with a phenol/chloroform(/isoamyl alcohol) separation treatment, to remove most of the hydrolyzed products.
- the DNA is then precipitated out of solution by the addition of alcohol.
- the unique process of DNA extraction of the present invention whereby hmwDNA can be isolated from a natural sample, comprises: preparing an aqueous suspension of the natural sample; gently emulsifying the suspension with an organic solvent; gently separating the aqueous, DNA- containing phase from the organic phase; and precipitating the DNA from the aqueous solution.
- the isolation process comprises additional steps of: gently resuspending the DNA and mixing the solution with a cationic detergent; re-emulsifying the solution with an organic solvent; separating the DNA-containing aqueous solution from the organic phase; reprecipitating the DNA; resuspending the DNA; and passing the suspension through a gradient, to separate out the hmwDNA.
- each step of reagent addition, suspension, mixing, and separation be performed gently (with deliberate care), to reduce or to avoid physical shearing of the sample DNA.
- all additions and separations to and from the sample are performed by a gentle means (e.g., pouring, or pipetting with a wide bore pipette tip).
- All mixing is preferably accomplished by a gentle means, whereby intermixing of solutions is accomplished with a minimal amount of solution turbulence (e.g., rocking, rolling, or rotating the solution mixture). Most preferably mixing comprises rotation of the sample.
- the separation of suspension and emulsion phases is preferably accomplished by centrifugation at a speed, and for a duration, sufficient to separate the phases.
- centrifugation is performed at least about 4000 rpm for at least about 10 min.
- the organic solvent of choice is phenol, and it is preferably warmed above normal room temperature; preferably to a temperature of at least about 35°C, and most preferably at least about 65°C.
- emulsion must be done gently, and should be done for a period of time sufficient to allow deproteinization.
- emulsion is performed for at least about 30 min. Although it is preferred that the phenol be warmed to a temperature above normal room temperature, the emulsion may be performed at room temperature.
- Precipitation of DNA from an aqueous solution is best performed by the addition of alcohol to the solution in a sufficient amount, followed by gentle mixing for a time sufficient to allow for complete precipitation of DNA.
- DNA precipitation step is accomplished by the addition of isopropanol, more preferably 0J vol isopropanol, and the solution rotated for at least about 30 min @ RT.
- precipitated DNA (the "DNA pellet” after centrifugation) may be washed one or more times with 70% ethanol.
- the practitioner may choose to treat the resuspended DNA, precipitated after the first organic solvent extraction, with a digestive enzyme.
- a digestive enzyme Many proteolytic enzymes are known in the art (e.g., Proteinase K).
- a further modified process may also include sample treatment with an anionic detergent, such as SDS. These treatments are well known in the art, but not an essential feature of the present invention, and are advantageously omitted to reduce the amount of sample handling.
- the DNA pellet is preferably resuspended into solution and treated with a cationic detergent.
- Cationic detergents have been shown not only to precipitate nucleic acids, but also to treat the biological sources of nucleic acid, lysing cells, and solubilizing contaminating lipids and proteins (Schneider, 1997).
- Commercially useful detergents are known in the art, and include cetrimonium compounds (such as cetyl pyridinium bromide and cetyltrimethylammonium bromide), and benzalkonium compounds (such as alkylbenzyldimethylammonium chlorides).
- the cationic detergent comprises cetyl trimethylammonium bromide (CTAB).
- the DNA solution is mixed with the cationic detergent for a time sufficient to allow separation of the nucleic acids from contaminating components.
- mixing comprises rotating the mixture for at least about 5 min. More preferably, the mixture is incubated at a temperature of at least about 65°C after an initial 5 min rotation, followed by an additional 5 min rotation.
- the mixture is preferably treated to a second emulsion with an organic solvent (preferably an organic solvent different from the organic solvent used for the first critical separation) to separate the nucleic acids from the detergent, other solvent residues, and remaining sample contaminants.
- the solvent is preferably chloroform, and treatment preferably comprises gentle mixing for at least about 30 min.
- Final emulsion separation of the DNA-containing aqueous phase from the organic phase preferably comprises centrifugation of the emulsion at least about 10,000 rpm for at least about 10 min.
- the DNA pellet may be resuspended into solution and prepared for gradient separation of the DNA on the basis of polymer size.
- Gradient separation may be accomplished using any of a variety of techniques well known in the art (e.g., electrophoresis, chromatography, density gradient separation).
- the gradient separation comprises a density gradient (such as cesium chloride gradients or sucrose gradients known in the art), and most preferably comprises passing the DNA solution through a sucrose gradient.
- hmwDNA is isolated, and can be removed from the gradient by fractionation or direct extraction.
- DNA libraries comprising hmwDNA extracted from natural samples will greatly facilitate analysis of the genetic diversity of the natural environment for both academic and commercial purposes.
- DNA libraries derived from natural samples provide an invaluable tool for research and development into novel biochemicals useful in a variety of applications (e.g., medical, industrial, commercial) as discussed earlier. It is an object of the present invention to provide a library of hmwDNA fragments isolated from a natural sample.
- the extracted DNA fragments are inserted into a cloning vector (including but not limited to expression vectors) of choice.
- a cloning vector including but not limited to expression vectors
- a variety of well known techniques are available to the practitioner for the successful incorporation of a DNA fragment into a vector (including, but not limited to, blunt end ligation, linker ligation, homopolymeric tailing, restriction digestion).
- the DNA insert-vector construct is stable and capable of replication of the DNA insert, to provide for long-term storage and amplification of the isolated genetic information.
- the cloning vector may possess any of a number of characteristics useful for genetic engineering.
- the vector of choice is an expression vector (i.e., a genetic construct wherein the DNA fragment is operably incorporated into a vector to allow transcription and translation of the DNA insert) expression regulatory regions (e.g., promoter regions and start codons) may be provided by either the vector DNA , the insert DNA, or separately inserted by the practitioner. It is a requirement of the present invention that the vector is capable of incorporating hmwDNA.
- a variety of genetic vectors may be used including, but not limited to; plasmids, cosmids, phagemids, modified viruses, shuttle vectors, and artificial chromosomes (e.g., YAC's and BAC's)(See Figure 2).
- Vectors may include other features useful in the manipulation and analysis of the DNA insert, including, but not limited to; DNA linkers, restriction nuclease sites, high copy origins of replication; insertion sequences; and indicator and/or selectable markers, the use of which are well known in the art.
- Vectors used in the present invention may be constructed by the practitioner, using techniques well known in the art, or may be commercially purchased (e.g., from Boehringer Mannheim Corp., Indianapolis, IN; Life Technologies, Inc., Rockville, MD; New England Biolabs, Inc., Beverley, MA; Pharmacia LKB Biotechnology, Inc., Piscataway, NJ; Stratagene, La Jolla, CA.).
- the vector libraries of the present invention can be stably introduced and maintained in a host organism for the purposes of DNA insert replication, and more preferably, DNA insert expression.
- the host organism may be any cell type from any living system: these include species from; Eubacteria, Archaebacteria, Protista, Plantae, Fungi, and Animalia. Recombinant host cell systems from each of these taxonomic Kingdoms, and a multiplicity of techniques for the incorporation of foreign DNA into different cell types are well known in the art; including, but not limited to; biolistic transfer; conjugation, electroporation, infection, liposome- mediated transfer, microinjection, protoplast fusion, transfection, and transformation.
- hmwDNA extraction from a natural sample is incorporated into an expression vector within a host cell capable of expressing one or more polypeptide(s) encoded by the hmwDNA insert.
- the multiplicity of novel polypeptides, and/or their biochemical products produced by expression library can then be screened for a chemical property or activity of interest. Standard protocols exist, and are obviously modified, for screening DNA libraries and the products produced therefrom for novel compounds of some desired chemical or biological characteristic.
- selection parameters are well known in the art, including but not limited to various biological selection regimes (e.g., cell or phage proliferation in the presence or absence of a compound, physiological marker systems), physical selection regimes (e.g., various cell sorting regimes), and chemical activity selection regimes (e.g., chromatographic separation).
- biological selection regimes e.g., cell or phage proliferation in the presence or absence of a compound, physiological marker systems
- physical selection regimes e.g., various cell sorting regimes
- chemical activity selection regimes e.g., chromatographic separation
- EXAMPLE 1 Recovery of High Molecular Weight DNA from a Natural Sample To demonstrate that hmwDNA can be isolated directly from a natural sample without the necessity of culturing, or otherwise pretreating, the genetic organisms contained within the sample, the methods of the present invention were applied to a natural soil sample taken from a local site.
- a 50 ml soil sample was suspended in a solution of 25mM Tris 8.0, 150mM NaCl, 25mM EDTA (Buffer I) to a final volume of 175 ml.
- 50 ml of equilibrated 65°C phenol was added to the soil suspension and emulsed by rotation (10-15 ⁇ m) for 30 min RT.
- the emulsion was centrifliged @ 4000 ⁇ m for 20 min.
- the aqueous phase was gently poured into a clean vessel, 0J vol isopropanol gently added to the aqueous solution (final volume -150 ml), and rotated for an additional 30 min RT.
- the mixture was centrifuged @ 4000 ⁇ m for 20 min and the supernatant discarded.
- the precipitated DNA "pellet” was further washed once with 70% ethanol and dried.
- DNA extract was resuspended in 6 mis of Buffer I plus 600 ⁇ l of 5M NaCl (i.e., gentle rotation for -10 min), and 6 ml of 65°C 2% CTAB (in 2M NaCl) was added. The mixture was rotated for 5 min, incubated @ 65°C for 10 min, and rotated an additional 5 min. Chloroform (6 ml) was added to the solution and rotated a further 30 min. The solution was then centrifuged @ 10,000 ⁇ m for 10 min.
- the aqueous phase was transferred into a clean vessel using a wide-bore 1 ml pipette, 0J vol isopropanol gently added to the aqueous solution, and rotated for 30 min RT.
- the DNA precipitate was allowed to settle to the bottom of the vessel, and the supernatant gently poured off.
- the DNA precipitate was washed repeatedly with 15 ml aliquots of 70% ethanol until the supernatant was clear. Final removal of ethanol was followed by resuspension of the DNA in 1 ml TE (the DNA precipitate was allowed to self-resuspend for -1 hr).
- the DNA solution was centrifuged @ 10,000 ⁇ m for 5 min, and any residual particulates removed.
- the DNA solution was loaded onto a 32 ml sucrose gradient (in l"x3.5" Ultraclear BeckmanTM centrifuge tube), comprising 8 ml steps of 20%, 30%, 40%, and 50% sucrose in TE.
- the gradient was ultracentrifuge @ 28,000 ⁇ m for 21 hrs (without braking).
- the gradient was eluted in 2 drop increments into a 96-well plate. Every other elution fraction was run on a pulse- field electrophoresis gel.
- DNA yield was approximately 1 microgram ( ⁇ g) per gram of soil, however the yield can vary depending on soil type (clay vs. sandy) as well as the location and time of sampling.
- EXAMPLE 2 Construction of a DNA Expression Library from a Natural Sample
- hmwDNA extracted directly from a natural sample can be inserted stably into an expression vector, whereby the compilation of individual recombinant vectors represents a library of diverse DNA fragments derived from that natural sample
- a DNA library was generated using hmwDNA extracted from a soil sample (as described in Example 1 above), inserted into a bacterial artificial chromosome (BAC), and transformed into E. coli.
- a composite hmwDNA library (-15,000 clones) in pBTP2 (a modified BAC -base vector) was created, comprised of 3 DNA sub-libraries. The construction was accomplished by digesting hmwDNA (separated by sucrose gradient centrifugation) with Hind III (see Fig. 1).
- the digested DNA was size purified by pulse field electrophoresis (DNA)(Bio-Rad, Hercules, CA). Three separate size ranges were excised; 50-100kbp, 100-150kbp and 150-200kbp fragments. The fragments were further purified on a second pulse field gel to remove residual small molecular weight DNA fragments trapped in the gel matrix, because smaller DNA fragments tend to ligate more efficiently than hmwDNA.
- the gel slices were dialysed against TE and digested with gelase (Bio-Rad) to dissolve the agarose. Ligation was performed using standard enzymes (T4 ligase and ATP) and 25 ng of vector at a 10: 1 vector to insert molar ratio. The ligation was drop dialysed with TE and transformed into E. coli (DH10B). Results of the library construct are provided in Table 1. Table 1. Library Results
- This 15,000 member library was constructed from a sub-sample of the soil-extracted hmwDNA. Extrapolating to the total 500ug sample obtained from a single 400g soil sample, total library constructs would approximate 10 clones.
- EXAMPLE 3 Diversity of a DNA library isolated from a Natural Sample
- FIG. 1 A sample of soil hmwDNA was subjected to PCR analysis using primers homologous to sequences found within small subunit (16S) rRNA.
- the use of rRNA sequences to determine species diversity and to establish phylogenetic relationships is well known in the art (for review see Hugenholtz, 1998).
- the use of degenerative oligos allows for the amplification of many different bacterial families. Over 200 16S DNA fragments were cloned and sequenced. The results indicate a wide diversity of sequences categorized within known families from around world. The majority of sequences, however, were unidentifiable bacterial families; presumably representing unknown bacterial strains.
- Figure 3 diagrams a phylogenetic tree based on sequences obtained from soil hmwDNA. Inco ⁇ orated into the diagram are bacterial representatives of divergent taxonomic families, including several from published reports of 16S subunit sequence analysis of DNA obtained from soil.
- hmwDNA expression libraries derived from a natural sample can be screened for some selected physiological characteristic, chemical or physical property, or biological activity
- a hmwDNA expression library derived from a soil sample was screened for a variety of activities (see Table 2).
- screening assays isolated a variety of clones from the soil hmwDNA library; e.g., clones capable of expressing known compounds such as indirubin and indole.
- the screen for antibacterial activity against a sensitive strain of Bacillus subtilis revealed three separate and independent antibacterial clones.
- An organic molecule, encoded by the soil DNA insert of this clone, has been identified as the source of the antibacterial activity. Sequence analysis of the soil hmwDNA insert fails to link the compound, or its source, to any currently known antibiotic or bacterial strain.
- EXAMPLE 5 Genetic and Chemical Analyses of a DNA Expression Library Isolate Obtained from a Natural Sample
- DNA isolates of a DNA expression library derived from a natural sample can genetically manipulated for detailed analysis of the DNA isolate and the polypeptide(s) it encodes
- one of the three antibacterial clones isolated and screened from Example 4 above (clone mg 1.1) was subcloned and further analyzed.
- Clone mg 1.1 insert size of 27 kb
- which produces a pu ⁇ le pigment exhibits antibacterial activity against B. Subtilis and S. aureus.
- the isolate was further analyzed.
- plasmid pTRANS -sacsB contains the T7-based transposon TRANS (derived from plasmid pGPSl, New England BioLabs, Beverly, MA), a ColEl origin of replication and a kanamycin resistance gene.
- Plasmid pTRANS-s ⁇ cB also encodes the Bacillus subtilis sacB gene in the vector portion of the plasmid, allowing for its counterselection in the presence of 5% sucrose.
- Transposition reactions were performed following the published protocol for pGPSl, followed by transformation of electrocompetent E. coli strain DH10B with 5 ⁇ l of the transposon reaction and selection of transformants on LB plates containing kanamycin (50 ⁇ g/ml), chloramphenicol (lO ⁇ g/ml), and sucrose (5%).
- the resulting transformants contained multicopy BAC plasmids with TRANS insertions.
- Transformants that lost the heterologous activity contain TRANS insertions in soil DNA sequences encoding that activity.
- plasmid DNA was isolated using the Qiagen Biorobot 9600 (Qiagen, Inc., Valencia, CA) according to the manufacturer's instructions, and sequenced using ABI Big Dye sequencing kit and run on an ABI 377 DNA sequencer. Bases were assigned using the Unix program Phred, the data was assembled using Phrap, and edited using Consed (University of Washington, Seattle, WA).
- Torsvik et al. “Comparison of phenotypic diversity and DNA heterogeneity in a population of soil bacteria", Appl. Environ. Micro. 56:782-787 (1990). Torsvik et al., In Ritz and Giller (eds.) Beyond the Biomass: Compositional and Functional Analysis of Soil Microbial Communities. John Wiley and Sons, Chichester (1994). Vining, "Functions of secondary metabolites", Annu. Rev. Microbiol. 44:395-427 (1990). Ward et al., "16S rRNA sequences reveal numerous uncultured microorganisms in a natural community", Nature 345:63-65 (1990). Yen et al., J Bacteriol. 173:5315-5327 (1991).
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NZ515641A NZ515641A (en) | 1999-06-02 | 2000-06-01 | Methods and compositions for isolating high molecular weight DNA from natural samples |
MXPA01012345A MXPA01012345A (en) | 1999-06-02 | 2000-06-01 | Methods and compositions for isolating high molecular weight dna from natural samples. |
IL14672800A IL146728A0 (en) | 1999-06-02 | 2000-06-01 | Methods and compositions for isolating high molecular weight dna from natural samples |
AU51787/00A AU778943B2 (en) | 1999-06-02 | 2000-06-01 | Methods and compositions for isolating high molecular weight DNA from natural samples |
EP00936472A EP1208229A4 (en) | 1999-06-02 | 2000-06-01 | Methods and compositions for isolating high molecular weight dna from natural samples |
BR0011249-6A BR0011249A (en) | 1999-06-02 | 2000-06-01 | Methods and compositions for high molecular weight DNA isolation from natural samples |
JP2001500817A JP2003520024A (en) | 1999-06-02 | 2000-06-01 | Methods and compositions for isolating high molecular weight DNA from natural samples |
CA002375873A CA2375873A1 (en) | 1999-06-02 | 2000-06-01 | Methods and compositions for isolating high molecular weight dna from natural samples |
KR1020017015469A KR20020034087A (en) | 1999-06-02 | 2000-06-01 | Methods and compositions for isolating high molecular weight DNA from natural samples |
NO20015730A NO20015730L (en) | 1999-06-02 | 2001-11-23 | Methods and Preparations for Isolating Hay Molecular DNA from Natural Samples |
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