Abstract:
A method for encoding and identifying biological materials is disclosed. The method may include encoding and identifying plants from which controlled substances may be derived and other materials for which movement and distribution may need to be tracked. The biological material may be first encoded using DNA oligomers. A spray method or the use of an encoded substrate, both using these DNA oligomers for encoding the biological material, may be employed. The biological material, or a part of the biological material, may be first encoded by atomizing a solution containing DNA oligomers onto it and then dried by an appropriate method. Thereafter, the part of the encoded biological material, or the nitrocellulose substrate, may be dissolved with a buffer solution for extracting the DNA oligomers. Then, the dissolved solution may be used for generating a barcode by a suitable detection scheme.

Description:
BACKGROUND 
       [0001]    1. Field of the Disclosure 
         [0002]    The present disclosure relates generally to biological encoding systems, and more particularly to DNA barcodes for distinguishing, tracking, and controlling biological material such as certain plants and seeds. 
         [0003]    2. Background Information 
         [0004]    Many agricultural products may need to be regulated for being authenticated, verified, tracked, and controlled to prevent the cultivation, manufacturing, distribution, and sale of unauthorized biological material products that are considered illegal. Some of these biological material products, from which controlled substances are derived, may include plants such as cannabis plants, coca plant, opium poppy, khat, and iboga, among others. Another, biological material product that may need to be regulated may include genetically-modified seeds which are protected by legally enforced plant-breeders and other intellectual property rights. 
         [0005]    A variety of plants and seeds may be confused with those plants and seeds that are used for medical roles in human society, or genetically-modified seeds that are authorized for being sold or resold by farmers. For example, according to the variety of cannabinoid content (active ingredients) and other compounds of cannabis plants, these can be classified as toxic or non-toxic for human consumption. Several cannabis plants with non-toxic cannabinoids can be used as a physician-recommended form of medicine or herbal therapy; however, some cannabis plants that may include toxic cannabinoids may cause negative effects, such as problems with memory and learning. These toxic cannabinoids may be considered illegal. 
         [0006]    There are few methods for identifying legal biological material products from illegal varieties; however, there are certain methods that may modify these biological material products or their production, which may be considered neither convenient nor accurate, and may represent a high cost for several regulation entities. For example, the use of genetic engineering may innately modify the plant in a very fundamental form. 
         [0007]    There is therefore a need to be able to distinguish authorized biological material products from common, illegal toxic varieties of biological material products; a new method may be applied to perform the identification of legal and illegal biological materials with more accuracy and lower cost. 
       SUMMARY 
       [0008]    According to various embodiments of the present disclosure, a method for encoding and identifying biological materials, such as plants and seeds, may be disclosed. This method may allow to encode and identify plants from which controlled substances, such as cocaine, heroin, and marijuana, may be derived. Furthermore, this method may be applied to encode and identify biological material for which movement and distribution may need to be controlled and tracked, such as genetically-modified seeds. 
         [0009]    In this disclosure, two different methods may be described for encoding biological materials. One method to encode biological materials may include using a spray method. The spray method may be performed by a dispensing device with a reservoir of a barcoded solution. This barcoded solution may include DNA oligomers combined with a suitable solution such as TE buffer (Tris EDTA pH 8). The barcoded solution may be in charge of encoding the biological material to be utilized in a later analysis. 
         [0010]    Another method to encode biological materials includes the use of an encoded substrate such as nitrocellulose. The nitrocellulose substrate may include DNA oligomers and may be wrapped around biological material when packaging. 
         [0011]    The samples obtained by the encoding methods may be soaked and dissolved in a buffer solution to extract the encoded DNA oligomers. This dissolved solution may be utilized for identifying the type of biological material by employing a suitable detection scheme. 
         [0012]    The type of encoded DNA oligomers detected by common detection schemes may be compared against a database to translate the meaning of the encoded DNA oligomer sequences. Additional information about the biological material may be obtained after having the detection results, including but not limited to plant breed, growth facility, lot number, and expiration date, among others. 
         [0013]    This method for distinguishing legal and illegal biological material may allow perform an accurate analysis and detection without altering biological material properties. 
         [0014]    In one embodiment, a method for encoding a biological material comprises forming a barcode solution including a sequence of DNA oligomers, wherein the sequence of DNA oligomers encodes information about the biological material; and atomizing the barcode solution on the biological material using a dispensing device. 
         [0015]    In another embodiment, a method for encoding a biological material comprises forming an encoded substrate including a nitrocellulose substrate encoded with a sequence of DNA oligomers, wherein the sequence of DNA oligomers encodes information about the biological material; drying at least a part of the biological material; and adhering the dried part of the biological material to the encoded substrate. 
         [0016]    In yet another embodiment, a method for encoding a biological material comprises forming an encoded substrate including a nitrocellulose substrate encoded with a sequence of DNA oligomers, wherein the sequence of DNA oligomers encodes information about the biological material; and wrapping the encoded substrate around a section of the biological material. 
         [0017]    In another embodiment, a method for distinguishing biological material comprises encoding biological material with a sequence of DNA oligomers, wherein the sequence of DNA oligomers forms encoded information about the biological material; dissolving encoded samples of the biological material in a buffer solution to extract the DNA oligomers from the encoded samples; detecting the DNA oligomers using a detection scheme to form a readout describing the sequence of DNA oligomers; and comparing the sequence of DNA oligomers to a database to translate the meaning of the sequence of DNA oligomers, wherein the DNA sequence describes encoded information about the biological material. 
         [0018]    Additional features and advantages of an embodiment will be set forth in the description which follows, and in part will be apparent from the description. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the exemplary embodiments in the written description and claims hereof as well as the appended drawings. 
         [0019]    It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0020]    The present disclosure can be better understood by referring to the following figures. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the disclosure. In the figures, reference numerals designate corresponding parts throughout the different views. 
           [0021]      FIGS. 1A to 1C  describe methods for encoding biological material using DNA oligomers.  FIG. 1A  describes a spray method for encoding biological materials using DNA oligomers, according to an embodiment.  FIG. 1B  describes an encoded substrate for encoding biological material with DNA oligomers, according to an embodiment.  FIG. 1C  shows an embodiment of an encoded substrate using a suitably-sized strip. 
           [0022]      FIG. 2  describes a method for extracting and detecting encoded DNA oligomers using samples obtained in  FIGS. 1A to 1C , according to an embodiment. 
           [0023]      FIG. 3  illustrates an encoding process that a pharmaceutical laboratory may follow to encode a stem of a medical cannabis plant, according to an exemplary embodiment. 
           [0024]      FIG. 4  illustrates an encoding process that an agricultural biotechnology corporation may follow to encode seeds that are genetically modified, according to an exemplary embodiment. 
           [0025]      FIG. 5  illustrates an encoding process that a pharmaceutical laboratory may follow to encode a leaf of a coca plant for medical purposes, according to an exemplary embodiment. 
       
    
    
     DETAILED DESCRIPTION 
       [0026]    The present disclosure is here described in detail with reference to embodiments illustrated in the drawings, which form a part here. Other embodiments may be used and/or other changes may be made without departing from the spirit or scope of the present disclosure. The illustrative embodiments described in the detailed description are not meant to be limiting of the subject matter presented here. 
       Definitions 
       [0027]    As used here the following terms may have the following definitions: 
         [0028]    “DNA oligomer” refers to a short single-stranded sequence of deoxyribonucleic acid (DNA) formed by bounded molecules. 
         [0029]    “Coding strand” refers to a synthetic short single-stranded sequence of DNA used to encode cannabis plants. 
         [0030]    “Barcode” refers to a pattern that allows the identification or verification of the type of a living being based on a DNA sequence. 
         [0031]    “Biological material” refers to substances containing genetic information from organisms of the Plantae kingdom, such as plants and seeds, capable of reproducing themselves or being reproduced in a biological system. 
       DESCRIPTION OF THE DRAWINGS 
       [0032]      FIGS. 1A to 1C  describe methods for encoding biological material using synthetic DNA oligomers. According to an embodiment, these encoding methods with DNA oligomers may allow to identify plants and seeds that are legal from illegal varieties.  FIG. 1A  describes an encoding method using a spray solution with DNA oligomers; and  FIG. 1B  describes an alternative encoding method using an encoded substrate with oligomers. However, these two methods may not intend to limit the disclosure, other methods may be applied to encode biological materials. 
         [0033]    The sequences of DNA oligomers used in  FIG. 1A  and  FIG. 1B  may be agreed upon by a standards committee. This standards committee may have an agreement and cooperation among different parties of interest such as law enforcement, distributors, manufacturers, pharmacies, end users, and others entities. 
         [0034]    Furthermore, the diverse types of DNA oligomers which may be used to encode each biological material may be according to specific information such as breed, lot number, growth facility, expiration date, and among others. 
         [0035]      FIG. 1A  describes a spray method  100  for encoding biological materials. In this embodiment, a plant  102 , such as a cannabis plant, may be encoded with synthetic DNA oligomers; nevertheless, this spray method  100  may also be used for other biological materials. 
         [0036]    In order to encode plants  102  using spray method  100 , particular types of DNA oligomers may be needed, as well as, a suitable solution, and a dispensing device  104 . 
         [0037]    The coding strands (CS  106 ) of DNA oligomers, which may be used to encode plant  102 , may preferably be between about 20 to about 50 base pairs in length. Each strand of each DNA oligomer may be at a concentration of at least about 1 μM to about 50 μM. Further, a minimum of about 100 picomols of each CS  106  may be deposited onto a detectable area of plant  102 , where this amount may be approximately from about 50 μL of 2 μM solution. 
         [0038]    The suitable solution utilized in spray method  100  may be appropriate for solubilizing DNA oligomers and avoiding problems such as degradation. This solution may be TE buffer (Tris EDTA pH 8) which must be freshly autoclaved. Alternatively, distilled water (dH2O) may be used. In the suitable solution, one or more DNA oligomers with characteristics described above may be included and deposited into a dispensing device  104 . The mixture of the suitable solution with DNA oligomers may produce a barcoded solution  108 . 
         [0039]    The dispensing device  104 , which may be employed in spray method  100  for depositing the barcoded solution  108  to plant  102 , may be capable of reproducibly depositing controllable quantities of the CS  106  from the barcoded solution  108 . 
         [0040]    The spray method  100  may be employed when a detectable part of plant  102  may be atomized with barcoded solution  108  using dispensing device  104 . Subsequently, the suitable atomized part of plant  102  may require to be dried for a long-term storage. This drying process may be performed by applying different methods such as exposing plant  102  to air in for a determined amount of time or using a desiccator device. The determined concentration covered with barcoded solution  108  may be used as a sample for later analysis. 
         [0041]      FIG. 1B  describes an encoded substrate  112  with DNA oligomers for encoding biological materials. A suitable substrate such as nitrocellulose substrate  114 , which may be encoded by CS  106  of DNA oligomers, may be used in this embodiment. Additionally, in this embodiment encoded substrate  112  may encode plant  102 ; however, this encoded substrate  112  may also be used for other biological materials. 
         [0042]    This encoding process may begin when a part of plant  102  may be first dried by different methods mentioned in  FIG. 1A . Subsequently, the dried part of plant  102  may be adhered into a surface of nitrocellulose substrate  114  encoded with DNA oligomers. 
         [0043]    Alternatively, in  FIG. 1C , a suitably-sized strip of nitrocellulose substrate  114  may be encoded with DNA oligomers. A suitable section of plant  102  may be adhered to or wrapped around the encoded suitably-sized strip at any point either during or after manufacture, prior to shipping plant  102  to a customer. 
         [0044]      FIG. 2  describes a DNA oligomer extraction and detection method  200  using samples obtained in  FIG. 1 . 
         [0045]    After encoding biological materials by methods mentioned in  FIG. 1 , the corresponding encoded samples may be analyzed by detecting and validating the encoded DNA oligomers of these biological materials. 
         [0046]    The encoded samples, such as the spray-encoded section of the plants  102  obtained in  FIG. 1A , and nitrocellulose substrate  114  used in  FIG. 1B , may be soaked and dissolved in an appropriate buffer solution  202 . This buffer solution  202  may be phosphate buffered saline (PBS), where the volume to be used may vary from about 0.1 mL to about 5 mL for an appropriate amount of time from about 30 seconds to about 3 minutes. Buffer solution  202  may extract the encoded DNA oligomers from the encoded samples of biological materials, which may be used to analyze these products. 
         [0047]    Subsequently, the dissolved solution obtained by the mixture of buffer solution  202  and DNA oligomers may be optionally filtered through a common 0.22 μm syringe filter. The syringe filter may remove unnecessary particles that may affect the detection of DNA oligomers during a decoding method. In another embodiment, a filter integrated into an assay device may be used to detect the type of biological material. 
         [0048]    Thereafter, the encoded DNA oligomers may be detected by common detection schemes, such as lateral flow assays, microarray detection, polymerase chain reaction (PCR), and solution-based Förster Resonance Energy Transfer (FRET) assays, among others. If all of the appropriately encoded DNA oligomers are present and detectable on the sample, then readout from the chosen detection system may be compared to a database to translate the meaning of the DNA oligomer sequences detected. 
         [0049]    The presence or absence of particular encoded DNA oligomer sequences may allow entities to distinguish, track, and control the biological material. When DNA oligomers are decoded, certain information about the biological material may be obtained, including plant breed, growth facility, lot number, and expiration date, among others. 
       EXAMPLES 
       [0050]    In Example #1,  FIG. 3  describes an encoding process  300  that a pharmaceutical laboratory may follow to encode a stem  302  of a medical cannabis plant. The encoding process  300  of stem  302  may be applied before packaging and selling it to a customer for accomplishing the regulatory medicinal controls. Stem  302  is sprayed  304  with about 50 μL to about 100 μL of a concentrated barcoded solution  108  containing oligomers that corresponds to a breed type, lot number, and expiration dates of cannabis plant, from where stem  302  was extracted. The barcoded solution  108  is deposited on the surface of stem  302  using a dispensing device  104  having a spray nozzle module. Then, the spray-encoded stem  302  may be dried  305  using a desiccator device  308 . Finally, stem  302  may be ready to be packaged  310  and sold to the customer. 
         [0051]    In Example #1,  FIG. 4  describes an encoding process  300  that an agricultural biotechnology corporation may follow to encode seeds  402  that are genetically modified. The encoding process  300  of seeds  402  may be applied before packaging and selling them to a customer for accomplishing the regulatory controls. Seeds  402  are sprayed  304  with about 50 μL to about 100 μL of a concentrated barcoded solution  108  containing oligomers that corresponds to a breed type, lot number, and expiration dates of seeds  402 . The barcoded solution  108  is deposited on the surface of seeds  402  using a dispensing device  104  having a spray nozzle module. Then, the spray-encoded seeds  402  may be dried  305  using a desiccator device  308 . Finally, seeds  402  may be ready to be packaged  310  and sold to the customer. 
         [0052]    In Example #1,  FIG. 5  describes an encoding process  300  that a pharmaceutical laboratory may follow to encode a leaf  502  of a coca plant for medical purposes. The encoding process  300  of leaf  502  may be applied before packaging and selling it to a customer for accomplishing the regulatory medicinal controls. Leaf  502  may be dried  305  using a desiccator device  308 . Finally, leaf  502  may be ready to be packaged  310  and sold to the customer. 
         [0053]    While various aspects and embodiments have been disclosed, other aspects and embodiments are contemplated. The various aspects and embodiments disclosed are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims. 
         [0054]    The embodiments described above are intended to be exemplary. One skilled in the art recognizes that numerous alternative components and embodiments that may be substituted for the particular examples described herein and still fall within the scope of the invention.