WO2016178895A1

WO2016178895A1 - Genetic analysis of commodities and raw materials

Info

Publication number: WO2016178895A1
Application number: PCT/US2016/029724
Authority: WO
Inventors: James A. Hayward; Michael E. Hogan; MingHwa Benjamin LIANG; Lawrence JUNG; Yuhua Sun
Original assignee: Apdn (B.V.I.) Inc.
Priority date: 2015-05-01
Filing date: 2016-04-28
Publication date: 2016-11-10

Abstract

Methods for genetic analysis of commodities and raw materials containing DNA, including components and ingredients of dietary supplements are disclosed. The method include extracting DNA from at least one component of a commodity, and the DNA is genetically analyzed by any process used in the art, such as next-generation sequencing methods, to identify at least one DNA sequence. The DNA sequence or a plurality of DNA sequences may then be used authenticate the commodity, or alternatively, to identify and or quantify one or more plant or animal species of the commodity and its components of a commodity or raw material. The one or more identified plant or animal species identified by the methods of the invention as components of a commodity or raw material may be used to verify security of supply chain by tracing a commodity throughout the chain and to compare the analyzed goods to specifications as purchased for compliance, standardization, expected monetary value, and/or accuracy.

Description

GENETIC ANALYSIS OF COMMODITIES AND RAW MATERIALS

Field of the Invention

The present invention pertains to a method for genetic analysis of commodities and raw materials having DNA, which may include materials such as herbs, botanical supplements and botanical remedies, vitamins, coffee, teas, cocoa, rice, grains, vegetables, fruits, fibers of animal derivation, fibers of plant derivation, agricultural products and additional such goods commonly sold or traded as commodities in the market place. More particularly, the present invention and the exemplary embodiments disclosed herein relate to genetic analysis of DNA of a commodity of interest to identify and/or verify the type of commodity.

Background

The purchase and sale of commodities and raw materials involve enormous sums of money annually, very large portions of global trading, as well as essential basis of commercial and governmental incomes. The financial impact of commodities trading on the world economy cannot be overstated, and therefore verifiable tracing and tracking of commodities is imperative, ideally on a source-to- shelf basis. Genetic analysis of commodities would provide the identification and verification of individual species throughout shipment routes and supply chains. The transport, exchange, and processing of commodities can be problematic since materials may be substituted for, or combined with, those of lesser quality or cost, potentially including materials that may pose performance issues or risks in the commodities or final products composed of those commodities.

For examples, herbs and botanicals intended for ingestion by animals, including humans, may easily be substituted or combined with other plant materials, and such changes would be very difficult to detect by a manufacturer and certainly more difficult for a consumer. While dietary supplements are generally manufactured according to good manufacturing standards, accurate and consistent sourcing, verifiable standardization of supplement ingredients such as herbs, vitamins and/or botanicals, and the tracing of those ingredients throughout the

manufacturing process are all imperative and sometimes problematic. Dietary supplement ingredients may be sourced and purchased from many locations across the globe, and are often subject to a myriad of restrictions, laws and international treaties, including CITES (Convention on International Trade in Endangered Species). Compliance with all applicable legal requirements is also imperative.

Providing and ensuring a verifiable and secure supply chain for commodities would greatly increase safety in the use of the individual commodities, and have a positive impact on commodity trading on a global basis by, inter alia, decreasing or entirely avoiding theft, ensuring that the quality, source, and intrinsic properties of a particular commodity are as expected and purchased, thereby improving safety and decreasing commercial risks and lost revenues.

An effective method of analyzing DNA will fulfill the need of verifying and securing the value, quality, and origin of commodities, and enhance consumer safety as well as commercial economic gains.

SUMMARY

The disclosed invention provides exemplary embodiments of methods for genetic analysis of commodities and raw materials containing DNA, including components and ingredients of dietary supplements. A method includes extracting DNA from at least one component of a commodity, and genetically analyzing the extracted DNA by any suitable method, such as for instance a next-generation sequencing method, to identify at least one DNA sequence. The DNA sequence or a plurality of DNA sequences may then be used to identify one or more plant or animal species of the commodity and its components.

According to an exemplary embodiment of the disclosed invention, the one or more identified DNA sequences may be used to determine the presence of one or more plant or animal species included in a commodity or raw material.

According to an exemplary embodiment of the present invention, the one or more identified DNA sequences may be used to determine the relative amounts of the plant or animal species included in a commodity or raw material.

According to an exemplary embodiment of the present invention, the one or more identified plant or animal species included in a commodity or raw material may be used to verify security of supply chain by tracing a commodity throughout the chain. An embodiment may be utilized to authenticate commodities by comparison the DNA analysis and identification results with authenticated commodities and purchased commodities of uncertain origin. In addition, an embodiment may be utilized to compare the analyzed commodities to the specifications as purchased for compliance, standardization, expected monetary value, and/or accuracy. According to an exemplary embodiment of the present invention, the one or more identified plant or animal species included in a commodity may be compared to an industry standard or specifications of the commodity for authentication or to determine whether the commodity has been adulterated with material or materials having a different DNA.

DETAILED DESCRIPTION

Exemplary embodiments of the present invention provide a method for genetic analysis and verification of commodities and raw materials having DNA, which may include materials such as herbs, botanicals, vitamins, coffee, teas, cocoa, rice, grains, vegetables, fruits, fibers of animal derivation, fibers of plant derivation, agricultural products and additional goods commonly sold or traded as raw materials or commodities in the market place (referred to hereinafter as "commodity" or "commodities"). The method includes providing at least one commodity of portion thereof, and extracting DNA from that commodity. The extracted DNA is analyzed by at least one sequencing method, such as next-generation sequencing methods, or a method practiced in the art, to identify at least one DNA sequence. The at least one DNA sequence is used to identify one or more plant or animal species included in a commodity.

Analysis of Commodities

An exemplary embodiment of the disclosed invention is set forth via an example using commonly sold items of dietary supplements. Generally, dietary supplements may be any supplement, such as a botanical supplement or botanical remedy. The dietary supplement may contain a vitamin, a mineral, algae, fungi, micro-organisms, amino acids, enzymes, metabolites, certain extracts, concentrates of materials, and/ or herbs in combination with many other components, and may take any suitable form, such as for instance, pills, gel-capsules, capsules, granules and powders to be used in any of a variety of ways, including in drinks, or in solids. A dietary supplement is generally ingested by, and may be administered in other manners to, animals, including humans to assist in or complement other nutrient intake. Dietary supplement components will often be ground or minced into small particles that are no longer easily or visibly indentified. Such components may easily be mistaken or combined during the many manufacturing processes, thereby potentially introducing an unknown and unmarked ingredient into a dietary supplement. Unknown components may pose health and safety risks as well as possible legal ramifications. The ability to identify components within dietary supplements by any of a number of DNA analysis methods such DNA sequencing, RFLP analysis, hybridization and PCR, will assist the industry in health and compliance matters and provide consumers with an enhanced level of quality assurance.

DNA Extraction

DNA of commodities, including dietary supplements may be separated via various methods used in the art, including extraction, isolation and purification. A variety of nucleic acid extraction solutions have been developed for extracting DNA from a sample of interest. See, for example, Sambrook et al. (Eds.) Molecular Cloning, Cold Spring Harbor Press, 1989; and Green, Michael R., and Joseph Sambrook. Molecular cloning: a laboratory manual. New York: Cold Spring Harbor Laboratory Press, 2012; both of which are incorporated herein by reference.

Extraction methods may include, for example, a detergent-mediated step; a proteinase treatment step; a phenol and/or chloroform extraction step; and/or an alcohol precipitation step. DNA extraction solutions may include an ethylene glycol-type reagent or an ethylene glycol derivative to increase the efficiency of DNA extraction. Other extraction methods utilize grinding and/or boiling the sample in water as at least a portion of the process. Further methods, including solvent-based systems and sonication, may also be utilized in conjunction with other extraction methods.

Exemplary Extraction Protocols

According to an exemplary embodiment of the present invention, DNA fragments may be captured by using one or more nanoparticle based capture methods. Various column methods have been developed for binding a biopolymer to a matrix with affinity for that biopolymer. The matrix may allow for bio molecule immobilization, separation from contaminating components, and concentration of biomolecules, such as DNA from a complex mixture. In this technology, sub-micron ceramic nanoparticles may be used to capture relatively small DNA fragments from a relatively large volume of biological extracts and then deliver them, via low speed

centrifugation into a small pellet which may then be used directly for PCR or Next- Generation Sequencing (NGS) library preparation. That technology may be used for capturing and analyzing relatively small amounts of cell-free DNA fragments in human serum (e.g., to detect tumors) and to capture and analyze the small amounts of fragmented DNA in water samples (e.g., to detect pathogen DNA). According to exemplary embodiments of the present invention nanoparticle capture technology may be used to capture and to analyze small, highly fragmented DNA fragments obtained in processed herbal extracts. Concentrated and purified DNA may be used for NGS based Ultra-Bar Coding. NGS technology is discussed in more detail below. The extraction of DNA using nanoparticle based capture methods is described in more detail in U.S. Patent No. 7,964,380.

ANALYSIS AND CLONING OF EUKARYOTIC GENOMIC DNA

1. Depending on the type of sample, carry out one of the following procedures as step 1.

Protocol I

Drop freshly excised tissue into liquid nitrogen in the stainless- steel cup of a Waring Blender. Blend at top speed until the tissue is ground to a powder. Allow the liquid nitrogen to evaporate, and add the powdered tissue little by little to approximately 10 volumes of extraction buffer (lOmM Tris · CI (pH 8.0), 0.1 mM EDTA (pH 8.0), 20 μg/ml pancreatic RNAase, 0.5% SDS) in a beaker. Allow the powder to spread over the surface of the extraction buffer, and then shake the beaker to submerge the material. When all of the material is in solution, transfer the solution to a 50-ml centrifuge tube, incubate for 1 hour at 37°C, and then proceed to step 2, below.

2. Add proteinase K to a final concentration of 100 μg/ml. Using a glass gently mix the enzyme into the viscous solution. Proteinase K is stored as a stock solution at a concentration of

20mg/ml in H₂0.

3. Place the suspension of lysed cells in a water bath for 3 hours at 50°C. Swirl the viscous solution periodically.

4. Cool the solution to room temperature, and, if necessary, pour the solution into a centrifuge tube. Add an equal volume of phenol equilibrated with 0.5 M Tris · CI (pH 8.0) and gently mix the two phases by slowly turning the tube end over end for 10 minutes. If the two phases have not formed an emulsion at this stage, place the tube on a roller apparatus for 1 hour. Separate the two phases by centrifugation at 5000g for 15 minutes at room temperature. The pH of the phenol is preferably approximately 8.0 to prevent DNA from becoming trapped at the interface between the organic and aqueous phases.

5. With a wide-bore pipette (0.3 cm-diameter orifice), transfer the viscous aqueous phase to a clean centrifuge tube and repeat the extraction with phenol twice. When transferring the aqueous phase, the DNA is preferably drawn into the pipette very slowly to avoid disturbing the material at the interface. If the DNA solution is so viscous that it cannot easily be drawn into a wide-bore pipette, use a long pipette attached to a water- suction vacuum pump to remove the organic phase. Make sure that the phenol is collected into traps and does not enter the water line. With the vacuum line closed, slowly lower the pipette to the bottom of the organic phase. Wait until the viscous thread of aqueous material detaches from the pipette, and then carefully open the vacuum line and gently withdraw all of the organic phase. Close the vacuum line and quickly withdraw the pipette through the aqueous phase. Immediately open the vacuum line to transfer the residual phenol into the trap. Centrifuge the DNA solution at 5000g for 20 minutes at room temperature. Protein and clots of DNA sediment to the bottom of the tube. Pour the DNA solution into a 50-ml centrifuge tube, leaving behind the protein and clots of DNA.

6. To isolate very-high-molecular-weight DNA (-200 kb): After the third extraction with phenol, dialyze the pooled aqueous phases at 4°C four times against 4 liters of a solution of 50mM Tris · CI (pH 8.0) lOmM EDTA (pH 8.0) until the OD₂₇o of the dialysate is less than 0.05. Allow room in the dialysis bag for the volume of the sample to increase 1.5 to 2.0-fold. Continue to step 7.

To isolate DNA whose size is 100-150 kb: After the third extraction with phenol, transfer the pooled aqueous phases to a fresh centrifuge tube and add 0.2 volume of 10 M ammonium acetate. Add 2 volumes of ethanol at room temperature and swirl the tube until the solution is thoroughly mixed. The DNA will rapidly form a precipitate that can usually be removed from the ethanolic solution with a pasteur pipette whose end has been sealed and shaped into a U. Most of the contaminating oligo-nucleotides are left behind. If the DNA precipitate becomes fragmented it can be collected by centrifugation at 5000g for 5 minutes at room temperature in a swinging-bucket rotor. Wash the DNA precipitate twice with 70% ethanol, and collect the DNA by centrifugation as described above. Remove as much as possible of the 70% ethanol, and store the pellet in an open tube at room temperature until the last visible traces of ethanol have evaporated. Do not allow the pellet of DNA to dry completely; otherwise, it will be very difficult to dissolve. Add 1 ml of TE (pH 8.0) for each ~ 5 x 10⁶ cells. Place the tube on a rocking platform and gently rock the solution until the DNA has completely dissolved. This usually takes 12-24 hours.

7. Measure the absorbance of the DNA at 260 nm and 280 nm. The ratio of A₂₆o to A₂₈o should be greater than 1.75. A lower ratio is an indication that significant amounts of protein remain in the preparation. In this case, add SDS to a concentration of 0.5% and then repeat steps 2-7. 8. Calculate the concentration of the DNA (a solution with an OD₂6o of 1 contains approximately 50 μg of DNA per milliliter), and analyze an aliquot by pulsed-field gel electrophoresis or by electrophoresis through a 0.3% agarose gel poured on a 1% agarose support. The DNA should be larger than 100 kb in size and should migrate more slowly than linear dimeric molecules of intact bacteriophage λ DNA. Store the DNA at 4°C.

Protocol II

This method, which is adapted from Bowtell (1987), is used to prepare DNA simultaneously from many different samples of cells or tissues.

1. Prepare cell suspensions (or frozen cell powders) as described in step 1 of protocol I.

2. Transfer the cell suspensions to centrifuge tubes, and add 7.5 volumes of lysis solution consisting of 6 M guanidine HC1 (M_r = 95.6), 0.1 M sodium acetate (pH 5.5).

If the DNA is to be extracted from tissues, add the frozen cell powders to approximately 7.5 volumes of lysis solution in beakers. Allow the powders to spread over the surface of the lysis solution, and then shake the beakers to submerge the material. When all the material is in solution, transfer the solution to centrifuge tubes.

3. Close the tops of the tubes and incubate for 1 hour at room temperature on a rocking platform.

4. Dispense 18 ml of ethanol at room temperature into each of a series of disposable 50-ml polypropylene centrifuge tubes. Using wide-bore pipettes, carefully layer the cell suspensions under the ethanol.

5. Recover the DNA from each tube by slowly stirring the interface between the cell lysate and the ethanol with a pasteur pipette whose end has been sealed and bent into a U shape. The DNA will adhere to the Pasteur pipette, forming a gelatinous mass. Continue stirring until the ethanol and the aqueous phase are thoroughly mixed.

6. Transfer each pasteur pipette, with its attached DNA, to a separate polypropylene tube containing 5ml of ethanol at room temperature. Leave the DNA submerged in the ethanol until all of the samples have been processed.

7. Remove each pipette, with its attached DNA, and allow as much ethanol as possible to drain away. By this stage, the DNA should have shrunk into a tightly packed, dehydrated mass, and it is often possible to remove most of the free ethanol by capillary action by touching the U-shaped end of the pipette to a stack of Kimwipes. Before all of the ethanol has evaporated from the DNA, transfer the pipette into a fresh polypropylene tube containing 5 ml of ethanol at room temperature.

8. When all of the samples have been processed, remove each pipette with its attached DNA, and remove as much ethanol as possible as described in step 7 above. Do not allow the pellet of DNA to dry completely, otherwise, it may be difficult to dissolve.

9. Transfer each pipette to a fresh polypropylene tube containing 1 ml of TE (pH 8.0). Allow the DNAs to rehydrate by storing the tubes overnight at 4°C.

10. By the next morning, the DNAs will have become highly gelatinous but will still be attached to their pipettes. Using fresh, sealed pasteur pipettes as scrapers, gently free the pellets of DNA from their pipettes. Discard the pipettes, leaving the DNAs floating in the TE. Close the tops of the tubes and incubate the DNAs at 4°C on a rocking platform until they are completely dissolved. This often takes 24-48 hours.

11. Analyze an aliquot by pulsed-field gel electrophoresis or by electrophoresis through a 0.3% agarose gel poured on a 1% agarose support. The DNA should be -80 kb in size and should migrate more slowly than monomers. Store the DNA at 4°C.

Additional Notes:

i. DNA made by this procedure may be contaminated with a small amount of RNA. It is therefore necessary to estimate the concentration of DNA in the final preparation either by fluorimetry or by gel electrophoresis and staining with ethidium bromide. If desired, the amount of contaminating RNA can be minimized by transferring the rehydrated pellet of DNA (step 10) to a fresh polypropylene tube containing 1 ml of TE (pH 8.0) before scraping it from the pasteur pipette. This is a hazardous procedure, since there is a risk that the DNA will slide off the pipette during transfer.

Partial Digestion of Eukaryotic DNA with Restriction Enzymes

A method by which DNA can be fragmented in random fashion irrespective of its base composition and sequence, is mechanical shearing. However, DNA prepared in this way requires several additional enzymatic manipulations (repair of termini, methylation, ligation to linkers, digestion of linkers) to generate cohesive termini compatible with those of the vectors used to generate genomic DNA libraries (Maniatis et al. 1978). One hand, partial digestion with restriction enzymes that recognize frequently occurring tetranucleotide sequences within eukaryotic DNA yields a population of fragments that is close to random and yet can be cloned directly.

Fragments of eukaryotic DNA suitable for the construction of genomic DNA libraries are prepared as follows: Carry out pilot experiments to establish conditions for partial digestion of eukaryotic DNA. Guided by the results of the pilot experiments, digest a large amount of eukaryotic DNA and purify fragments of the desired size by density gradient centrifugation. Pilot Experiments:

1. Dilute 30 μg of high-molecular- weight eukaryotic DNA ( > 200 kb; see Protocol I) to 900μ1 with 10 mM Tris · CI (pH 8.0) and add 100 μΐ of the appropriate lOx restriction enzyme buffer.

If the concentration of the high-molecular-weight DNA is low, increase the volume of the pilot reactions and concentrate the DNA after digestion by precipitation with ethanol. Each pilot reaction should contain at least 1 μg of DNA to allow the heterogeneous products of digestion to be detected by staining with ethidium bromide. Handle the eukaryotic DNA carefully by using either pipette tips that have been cut off with a sterile razor blade to enlarge the orifice or disposable wide-bore glass capillaries. Make sure that the DNA is dispersed homogeneously throughout the buffer used for digestion. The chief problem encountered during digestion of high-molecular-weight DNA is unevenness of digestion caused by variations in the local concentration of DNA. Clumps of DNA are relatively inaccessible to restriction enzymes and can be digested only from the outside. Unless the DNA is evenly dispersed, the rate of digestion cannot be predicted or controlled. To ensure homogeneous dispersion of the DNA:

a. Allow the DNA to stand at 4°C for several hours after dilution and addition of lOx restriction enzyme buffer.

b. Gently stir the DNA solution from time to time using a sealed glass capillary.

c. After addition of the restriction enzyme, gently stir the solution for 2-3 minutes at 4°C before warming the reaction to the appropriate temperature.

d. After digestion for 15-30 minutes, add a second aliquot of restriction enzyme and stir the reaction as described above.

2. Carry out test digestions on aliquots of the batch of diluted DNA that will be used to prepare fragments for cloning. The amount of enzyme necessary will vary for each batch of enzyme and preparation of DNA. a. Using a wide-bore glass capillary or a cut-off disposable plastic pipette tip, transfer 60 μΐ of the DNA solution to a microfuge tube (tube 1). Transfer 30 μΐ of the DNA solution to each of nine additional labeled microfuge tubes. Stand the tubes on ice.

b. Add 2 units of the appropriate restriction enzyme to the first tube. Use a sealed glass capillary to mix the restriction enzyme with the DNA. Do not allow the temperature of the reaction to rise above 4°C. Using a fresh pipette tip, transfer 30 μΐ of the reaction to the next tube in the series. Mix as before, and continue transferring the reaction to successive tubes. Do not add anything to the tenth tube (no enzyme control), but discard 30 μΐ from the ninth tube. Incubate the reactions for 1 hour at 37°C.

c. At the end of the digestion, heat the reactions to 70°C for 15 minutes to inactivate the restriction enzyme. After cooling the reactions to room temperature, add the appropriate amount of gel-loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 30% glycerol in water). Mix the solutions gently, using a sealed glass capillary. Use a cut-off, disposable plastic pipette tip or a disposable wide-bore glass capillary to transfer the solutions to the wells of a

0.3. agarose gel poured on a 1% agarose support. Compare the size of the digested eukaryotic DNA with that of oligomers of bacteriophage λ DNA and plasmids (see notes to step 1).

Large scale Preparation of Partially Digested DNA

1. After conditions have been established in pilot experiments (see above), digest 100 μg of high- molecular- weight DNA with the appropriate amount of restriction enzyme for the appropriate time. To ensure that the conditions for the large-scale digestion are as identical as possible to those used in the pilot experiment, we prefer to set up replicas of the successful pilot reaction rather than a single large-scale reaction. In addition, if sufficient eukaryotic DNA is available, we recommend using three different concentrations of restriction enzyme that straddle the optimal concentration determined in the pilot experiment. At the end of the digestion, analyze an aliquot of the DNA in each digestion by gel electro-phoresis to make sure that the digestion has worked according to prediction. Until the results are available, store the remainder of the sample at O°C.

2. Gently extract the digested DNA twice with phenokchloroform. Precipitate the DNA with 2 volumes of ethanol at 0°C and redissolve it in 200 μΐ of TE (pH 8.0).

3. Prepare a 10-40% continuous sucrose density gradient in a Beckman SW40 polyallomer tube (or its equivalent). The sucrose solutions are made in a buffer containing 10 mM Tris · CI (pH 8.0), 10 mM NaCl, 1 mM EDTA (pH 8.0). Heat the DNA sample for 10 minutes at 68°C, cool it to 20°C, and load it onto the gradient. Centrifuge at 22,000 rpm for 22 hours at 20°C in a Beckman SW40 rotor (or its equivalent).

4. Using a 19-gauge or like needle, puncture the bottom of the tube and collect 350-μ1 fractions.

5. Mix 10 μΐ of every other fraction with 10 μΐ of water and 5 μΐ of gel-loading buffer I (0.25% bromophenol blue, 0.25% xylene cyanol FF, 40% (w/v) sucrose in water). Analyze the size of the DNA in each fraction by electrophoresis through a 0.5% agarose gel, using oligomers of plasmid DNA as markers. Be sure to adjust the sucrose and salt concentrations of the markers to correspond to those of the samples.

6. Following electrophoresis, pool the gradient fractions containing DNA fragments of the desired size. Dialyze the pooled fractions against 2 liters of TE (pH 8.0) at 4°C for 12-16 hours, with a change of buffer after 4-6 hours. Leave space in the dialysis bag for the sample to expand two-to threefold. Alternatively, if the volume of the pooled sample is sufficiently small, the DNA can be precipitated with ethanol without prior dialysis after first diluting the sample with TE (pH 8.0) so that the concentration of sucrose is reduced to below 10%.

7. Extract the dialyzed DNA several times with an equal volume of 2-butanol until the volume is reduced to about 1 ml. Add 10 M ammonium acetate to a final concentration of 2 M and precipitate the DNA with 2 volumes of ethanol at room temperature.

8. Dissolve the DNA in TE (pH 8.0) at a concentration of 300-500 μg/ml. Analyze an aliquot of the DNA (0.5 μg) by electrophoresis through a 0.5% agarose gel to check that the size distribution of the digestion products is correct.

Large Scale Preparation of Partially digested DNA

1. After conditions have been established in pilot experiments (see above), digest 100 pg of high-molecular-weight DNA with the appropriate amount of restriction enzyme for the appropriate time. To ensure that the conditions for the large-scale digestion are as identical as possible to those used in the pilot experiment, we prefer to set up replicas of the successful pilot reaction rather than a single large-scale reaction. In addition, if sufficient eukaryotic DNA is available, we recommend using three different concentrations of restriction enzyme that straddle the optimal concentration determined in the pilot experiment. At the end of the digestion, analyze an aliquot of the DNA in each digestion by gel electrophoresis to make sure that the digestion has worked according to prediction. Until the results are available, store the remainder of the sample at 0°C.

2. Gently extract the digested DNA twice with phenol chloroform. Precipitate the DNA with 2 volumes of ethanol at 0°C and redissolve it in 200 μΐ of TE (pH 8.0).

3. Prepare a 10-40% continuous sucrose density gradient in a Beckman SW40 polyallomer tube (or its equivalent). The sucrose solutions are made in a buffer containing 10 mM Tris · CI (pH8.0), 10 mM NaCl, 1 mM EDTA (pH 8.0). Heat the DNA sample for 10 minutes at 68°C, cool it to 20°C, and load it onto the gradient. Centrifuge at 22,000 rpm for 22 hours at 20°C in a Beckman SW40 rotor (or its equivalent).

4. Using a 19-gauge or like needle, puncture the bottom of the tube and collect 350-pl fractions.

6. Following electrophoresis, pool the gradient fractions containing DNA fragments of the desired size (e.g., 35-45 kb for construction of libraries in cosmids; 20-25 kb for construction of libraries in bacteriophage λ vectors such as EMBL3 and 4). Dialyze the pooled fractions against 2 liters of TE (pH 8.0) at 4°C for 12-16 hours, with a change of buffer after 4-6 hours. Leave space in the dialysis bag for the sample to expand two to threefold. Alternatively, if the volume of the pooled sample is sufficiently small, the DNA can be precipitated with ethanol without prior dialysis after first diluting the sample with TE (pH 8.0) so that the concentration of sucrose is reduced to below 10%.

DNA Analysis by Next-Generation Sequencing

DNA sequences of a commodity components or plant or animal components of dietary supplements may be sequenced and/or detected by, inter alia, a next-generating sequencing (NGS) technology. NGS technology is a category of high-throughput sequencing technologies (e.g., massively parallel sequencing), which may be used to identify the nucleic acid sequences of nuclear, mitochondrial, ribosomal and/or chloroplast DNA extracted from one or more commodity or dietary supplements. NGS technology may be used to accurately sequence relatively large nucleic acid sequences or an entire genome of an organism. In NGS, a plurality of relatively small nucleic acid sequences (e.g. DNA sequences) may be sequenced simultaneously from a DNA sample and a library of small segments (i.e., reads) may be compiled. The individual reads may then be reassembled to provide the sequence of a larger nucleic acid (e.g. DNA) sequence or a complete nucleic acid sequence. For example and without limitation, in NGS technology 500,000 sequencing operations may be run in parallel.

According to exemplary embodiments of the present invention, NGS may be used to completely sequence substantially the entire genome of a number of different plant or animal species included in a single component of a commodity. For example, a dietary supplement including a mixture of components (e.g., a mixture of ginko, st. john's wort, ginseng, Echinacea and saw palmetto) may be analyzed by NGS to sequence substantially the entire genomes of each species included in the supplement. For example, the chloroplast DNA for each species of ginko, st. john's wort, ginseng, Echinacea and saw palmetto included in the dietary supplement may be simultaneously analyzed by NGS. According to exemplary embodiments of the present invention, 100 product samples may be run at one time.

One example of NGS technology is to employ MALBAC followed by traditional PCR. MALBAC refers to Multiple Annealing and Looping Based Amplification Cycles. MALBAC may be used to amplify substantially a whole genome. MALBAC operates in quasi-linear fashion and may be used for single cell, whole genome amplification. In MALBAC, amplicons may have complementary ends. The complementary ends may form loops, which may prevent exponential amplicon copying, thus preventing amplification bias. MALBAC is discussed in more detail in Zong, Chenghang, et al. "Genome-wide detection of single-nucleotide and copy- number variations of a single human cell" Science 338.6114 (2012): 1622-1626. NGS is discussed generally, and in more detail in Mardis, Elaine R. "The impact of next-generation sequencing technology on genetics" Trends in genetics 24.3 (2008): 133-141; and Metzker, Michael L. "Sequencing technologies— the next generation" Nature Reviews Genetics 11.1 (2009): 31-46.

Polony Sequencing is an example of NGS technology. In Polony sequencing, millions of immobilized DNA sequences are read in parallel. Polony sequencing is an example of a multiplex sequencing technique in which numerous analytes are measured in a single run/cycle or a single assay. Polony sequencing has been found to be extremely accurate with a relatively low error rate. Polony Sequencing methods are discussed in more detail in Shendure, Jay, et al. "Advanced sequencing technologies: methods and goals" Nature Reviews Genetics 5.5 (2004): 335-344; and Shendure, Jay, and Hanlee Ji. "Next-generation DNA sequencing" Nature biotechnology 26.10 (2008): 1135-1145.

Massively Parallel Signature Sequencing (MPSS) is an example of NGS technology. MPSS can be utilized to identify and/or quantify mRNA transcripts in a sample. MPSS may be used to identify mRNA transcripts by generating 17-20 base pair signature sequences. MPSS methods are discussed in more detail in Brenner, Sydney, et al. "Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays" Nature biotechnology 18.6 (2000): 630-634.

Illumina Sequencing is an example of NGS technology in which DNA molecules and primers may be immobilized on a slide. The immobilized DNA molecules may be amplified by a polymerase to form DNA colonies (i.e., DNA clusters). Illumina Sequencing methods are discussed in more detail in Hanlee Ji. "Next-generation DNA sequencing" Nature biotechnology 26.10 (2008): 1135-1145; and Meyer, Matthias, and Martin Kircher. "Illumina sequencing library preparation for highly multiplexed target capture and sequencing" Cold Spring Harbor Protocols 2010.6 (2010): pdb-prot5448.

Pyrosequencing is an exemplary NGS technology. In Pyrosequencing, a luciferase may be employed to detect individual nucleotides added to a nascent DNA. Pyrosequencing amplifies DNA contained in droplets of water. The droplets of water may be immersed in an oil solution. Each droplet of water may include a single DNA template attached to a primer-coated bead. Pyrosequencing methods are discussed in more detail in Vera, J. Cristobal, et al. "Rapid transcriptome characterization for a nonmodel organism using 454 pyrosequencing" Molecular ecology 17.7 (2008): 1636-1647; and Ronaghi, Mostafa. "Pyrosequencing sheds light on DNA sequencing" Genome research 11.1 (2001): 3-11.

Oligonucleotide Ligation and Detection (SOLiD Sequencing) is an example of NGS technology. According to SOLiD sequencing, thousands of relatively small sequence reads (i.e., DNA fragments) may be simultaneously generated. SOLiD sequencing may also be referred to as a sequencing by ligation method. Sequence reads may be immobilized on a solid support for sequencing. SOLiD sequencing methods are discussed in more detail in Hanlee Ji. "Next- generation DNA sequencing" Nature biotechnology 26.10 (2008): 1135-1145; and Meyer, Matthias, and Ansorge, Wilhelm J. "Next-generation DNA sequencing techniques" New biotechnology 25.4 (2009): 195-203.

Ion Torrent Semiconductor Sequencing is an example of NGS technology. In Ion Torrent Semiconductor Sequencing, hydrogen ions are released and detected during DNA

polymerization. Ion Torrent Semiconductor Sequencing may also be referred to as a sequence- by-synthesis method. A deoxyribonucleotide triphosphate (dNTP) may be provided into a microwell. The microwell may hold a template DNA strand. If the dNTP is complementary to a leading template nucleotide, the dNTP may be incorporated into the complementary DNA strand and a hydrogen ion, which may be detected, will be released. Ion Torrent Semiconductor Sequencing methods are discussed in more detail in Quail, Michael A., et al. "A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers" BMC genomics 13.1 (2012): 341.

Heliscope Single Molecule Sequencing is an example of NGS technology. Heliscope Single Molecule Sequencing does not require PCR amplification. Heliscope Single Molecule Sequencing is a direct-sequencing method in which DNA may be sheared, tailed with a poly-A tail and then hybridized to a surface of a flow cell. A relatively large numbers of molecules (e.g., billions of nucleotides) may be sequenced in parallel. Heliscope Single Molecule Sequencing methods are discussed in more detail in Pushkarev, Dmitry, Norma F. Neff, and Stephen R. Quake. "Single-molecule sequencing of an individual human genome" Nature biotechnology 27.9 (2009): 847-850.

DNA Nanoball Sequencing is an example of NGS technology. In DNA Nanoball Sequencing, relatively small fragments of DNA may be amplified using rolling circle replication to form DNA nanoballs. Amplified DNA sequences are ligated through the use of fluorescent probes, which may be used as guides. DNA Nanoball Sequencing methods are discussed in more detail in Ansorge, Wilhelm J. "Next-generation DNA sequencing techniques" New biotechnology 25.4 (2009): 195-203, and Drmanac, Radoje, et al. "Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays" Science 327.5961 (2010): 78- 81.

Single Molecule Real Time (SMRT) Sequencing is another example of NGS technology. In SMRT sequencing, DNA may be synthesized in relatively small containers, which may be referred to as zero-mode wave-guides (ZMWs). Unmodified polymerases may be attached to bottoms of the ZMWs. The unmodified polymerases may be used to sequence the DNA along with fluorescently labeled nucleotides. The fluorescently labeled nucleotides may be allowed to flow freely in the solution. Fluorescent labels may be released from each of the nucleotides as the nucleotides are incorporated into a DNA strand. SMRT sequencing is another example of a sequencing-by-synthesis method. SMRT Sequencing methods are discussed in more detail in Flusberg, Benjamin A., et al. "Direct detection of DNA methylation during single-molecule, realtime sequencing" Nature methods 7.6 (2010): 461-465.

According to an exemplary embodiment of the present invention, the one or more identified DNA sequences may be used to determine the presence of one or more plant or animal species included in the dietary supplement. Substantially all DNA included in the dietary supplement may be extracted and sequenced. The extracted DNA may include nuclear, mitochondrial, chloroplast and/or ribosomal DNA. Nucleic acids such as RNA may also be extracted and sequenced.

While NGS technology may be used to sequence substantially an entire genome of an identified species of plant or animal included in a dietary supplement, NGS may also be used to determine partial nucleic acid sequences (e.g., DNA sequences) of extracted DNA. For example, botanical DNA from a plant material included in the dietary supplement may be partially degraded, damaged or incomplete. Plant or animal DNA that is partially degraded or incomplete may vary in the degree of degradation between dietary supplements. The degree of degradation may vary as a result of, for example, manufacturing procedures of the dietary supplement. For example, a particular dietary supplement may have a particular botanical component from a particular botanical source which is almost fully degraded in one type of dietary supplement, but which is almost fully intact in a second type of dietary supplement. Different fragments of different lengths may be sufficiently intact in one dietary supplement, but might not be sufficiently intact in another dietary supplement. By extracting and sequencing the sequenceable DNA that is sufficiently intact in a particular dietary supplement, plant or animal species included in the dietary supplement may be determined.

According to an exemplary embodiment of the disclosed invention, DNA from a plant cell included in a dietary supplement may be degraded into many small fragments of DNA (e.g., 1,000,000 small fragments). The small fragments may be, for example, approximately 100- 300bp in length. The small fragments may be present in relatively low density (e.g., 1 part per million ( pM) relative to non-nucleic acid contents of the dietary supplement). All of the small fragments may be analyzed in "lots" of about 50,000 DNA fragments at a time. Thus, approximately 20 independent NGS sequencing reactions may be performed in parallel and all small fragments of DNA may be simultaneously sequenced. The sequenced fragments may all be compared to known sequences for identification of the plant species included in the dietary supplement. An analogous procedure may be applied to animal contents included in the dietary supplement. An analogous procedure may also be applied to any commodity that includes a biological component containing DNA.

Species Identification

According to exemplary embodiments of the present invention, DNA sequences may be compared to known DNA sequences (e.g. reference sequences) to determine the identity of one or more plant or animal species, which may, for example, be included in the dietary supplement. For example, all known nuclear, chloroplast and mitochondrial DNA sequences for all known plant and animal species may be stored in a single database. When a DNA sequence is identified according to exemplary embodiments of the present invention, the identified DNA sequence may be compared to the database of known DNA sequences to identify a plant or animal species associated with the identified DNA sequence. Partial DNA sequences may be sufficient for identifying one or more species. DNA sequences (e.g., partial DNA sequences) which are not found in the database of known DNA sequences may be stored and compiled into a database of new or unknown DNA sequences. Both known and new/unknown DNA sequences may be evaluated by bioinformatics analysis according to exemplary embodiments of the present invention.

Sequence polymorphisms may exist between related species, which may be used to differentiate one species from another species. For example, the cotton species G. barbadense and G.hirsutum include a number of sequence length polymorphisms between the chloroplast DNA of these species. The sequence length polymorphisms may be used to identify the presence of and to distinguish between G. barbadense and G.hirsutum derived material included in the commodity.

A sequence polymorphism between species may include a variable region between species, including for example, one or more single nucleotide polymorphisms (SNPs). The sequence polymorphism may include a sequence length polymorphism. The sequence polymorphism may include one or more nucleotide insertions or deletions. The variable region may include a sequence length polymorphism between the first cotton species and the second cotton species. For example and without limitation, the sequence length polymorphism may include one or more short tandem repeats (STRs). The variable region may include one or more microsatellites, which are also referred to as simple sequence repeats (SSRs).

According to exemplary embodiments of the present invention, plant species included in the dietary supplement may be identified according to chloroplast DNA sequences. Animal species may be identified according to mitochondrial DNA sequences.

Plant and animal contents of commodities may include highly degraded DNA. For example, plant/botanical contents of dietary supplements may be prepared from dried leaves or roots (e.g., by crushing or pulverizing the dried leaves or roots) which may be processed in alcohol to produce a tincature or heated in water to form a tea, and may then be freeze dried to produce a solid formulation for use in tablets, powders, capsules or mixtures. The preparation process may degrade DNA in the components or contents. Thus, the ability to isolate and sequence partial, and relatively small, fragments of DNA according to exemplary embodiments of the present invention may allow practical species identification using the contents of commercially available dietary supplements. DNA, including partial or relatively small strands of DNA, may also include detectable strands of DNA that are present in relatively small amounts. Thus, amplification and identification of relatively small amounts of DNA may be required for species identification according to exemplary embodiments of the present invention.

Bioinformatics Analysis of DNA sequences and "Bar Code" Database Development

According to exemplary embodiments of the present invention, substantially all sufficiently intact and sequenceable nuclear, mitochondrial, chloroplast or ribosomal DNA strands included in a commodity may be sequenced and a library of DNA sequences may be compiled (e.g., in a database). The library of DNA sequences may include relatively short DNA fragments, as described in more detail above. Standard bioinformatics procedures may be applied to the library of DNA sequences to determine one or more nuclear, mitochondrial, chloroplast or ribosomal DNA sequences which are specific to a particular plant or animal species included in a particular dietary supplement. Each identified DNA sequence which is specific to a particular plan or animal species, and is found to be sufficiently intact in a particular dietary supplement, may be used as a "bar code" for species identification. The bar codes may be employed for rapid species identification in subsequently tested dietary supplements to determine the presence or absence of one or more species of interest in a particular commodity.

According to an exemplary embodiment of the present invention, the one or more identified species included in the commodity may be compared to the specification of that material to determine whether the commodity meets anticipated parameters or may have been adulterated. For example, a commodity may include components or content derived of a different, unanticipated species, or a lower grade or quality of components. Thus, the

commodity may have lower efficacy or may be unsuitable for commercial use or an intended product. An adulterated commodity may also pose safety, health and legal risks to an

unsuspecting merchant or consumer.

According to exemplary embodiments of the present invention, rapidly identifiable DNA sequences particular to individual plant and animal species included in a commodity may be used for rapid species identification. For example, DNA sequences (e.g., short DNA fragments) that are particular to a single plant or animal species and that are identifiably intact in a particular commodity may be rapidly detected to identify the presence of the particular plant or animal species in the commodity. Rapid detection of plant or animal species may be performed in-field (e.g., by using rapid PCR, isothermal amplification and detection or microarray methods).

According to exemplary embodiments of the present invention, the detection of the constituent plant or animal species in the commodity may be performed quantitatively to determine relative plant or animal content concentrations in a particular commodity.

According to an exemplary embodiment of the present invention, the one or more identified plant or animal species included in the commodity may be compared to the commodity specifications to determine whether the components are proper.

The disclosures of each of the references, patents and published patent applications disclosed herein are each hereby incorporated by reference herein in their entireties.

In the event of a conflict between a definition herein and a definition incorporated by reference, the definition provided herein is intended.

Having described exemplary embodiments of the present invention, it is further noted that it is readily apparent to those of ordinary skill in the art that various modifications may be made without departing from the spirit and scope of the present invention.

Claims

We claim:

1. A method for genetic analysis of a commodity including DNA, the method comprising: extracting DNA from at least one component of the commodity;

genetically analyzing the extracted DNA;

identifying the extracted DNA as belonging to one or more plant species, and/or one or more animal species.

2. The method according to claim 1, wherein the extracted DNA is analyzed by a DNA analysis method selected from the group consisting of DNA sequencing, RFLP analysis, hybridization and PCR.

3. The method according to claim 1, wherein the DNA analysis method is a massively parallel (next generation) DNA sequencing method.

4. The method according to claim 2, wherein DNA analysis method identifies DNA from a single animal species or a single plant species.

5. The method according to claim 2, wherein DNA analysis method identifies DNA from a plurality of animal species.

6. The method according to claim 2, wherein DNA analysis method identifies DNA from a plurality of plant species.

7. The method according to claim 1, wherein the commodity is a dietary supplement.

8. The method according to claim 7, wherein the dietary supplement is selected from the group consisting of an herb, a botanical supplement and a botanical remedy.

9. The method according to claim 1, wherein the commodity is selected from the group consisting of coffee, tea, cocoa, rice, a grain, a vegetable, a fruit, a plant fiber and an animal fiber.

10. The method according to claim 2, wherein the DNA analysis method provides the relative amounts of to one or more plant species, and/or one or more animal species in the commodity.

11. A method for determination of the level of purity of a commodity containing DNA, the method comprising:

extracting DNA from at least one component of the commodity;

genetically analyzing the extracted DNA;

identifying the extracted DNA as belonging to one or more plant species, and/or one or more animal species as the DNA species profile of the extracted DNA;

comparing the DNA species profile of the extracted DNA with a standard DNA species profile of the authentic commodity; and

thereby determining the level of purity of the commodity.

12. The method according to claim 11, wherein the extracted DNA is analyzed by a DNA analysis method selected from the group consisting of DNA sequencing, RFLP analysis, hybridization and PCR.

13. The method according to claim 11, wherein the DNA analysis method is a DNA sequencing method is massively parallel (next generation) DNA sequencing.

14. The method according to claim 12, wherein DNA analysis method identifies DNA from a single animal species or a single plant species and thereby authenticates the purity of the commodity.

15. The method according to claim 11, wherein the commodity is a dietary supplement.