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Until recently, forensic botany has been an under utilized resource for linking seized plant evidence to criminals, victims and crime scenes. Forensic botany has not been used much primarily because of lack of knowledge among crime scene investigators, police and prosecutors (Bock and Norris 1997). Botanical evidence can be instrumental in a wide variety of investigations, including homicides, kidnappings, rapes, and drug smuggling and distribution operations.
Drug smuggling and trafficking are a serious problem in the United States. In 2008 alone, the Department of Homeland Security (DHS) and the Drug Enforcement Administration (DEA) seized a combined 1,144,374 kilograms of marijuana, 103,795 kilograms of cocaine and 1,653 kilograms of heroin (personal communication, Matthew Stentz, DHS-ICE, USDOJ 2009). These figures only represent the tip of the iceberg, and do not include other federal, state or local agency seizures, or drugs that were not detected and subsequently seized by agents.
Significant quantities of marijuana (primarily Cannabis sativa) are smuggled into the United States from Mexico and Canada. One of the primary directives of the DHS and DEA is to make every attempt to identify, disrupt and dismantle drug smuggling organizations. Once these organizations are identified, it is up to the criminal investigator to develop sufficient probable cause to arrest and prosecute individuals that are associated with this illicit trade. Agents and investigators are also responsible for linking distributors, suppliers and transporters to prove drug smuggling/trafficking conspiracies. Once agents obtain enough probable cause to arrest suspects and search and seize evidence and properties, the suspects are subsequently charged with violations of United States law, to wit, Title 18 § 371 (conspiracy), Title 21 § 841(a)(1) and Title 21 § 846 (conspiracy to possess with intent to distribute a controlled substance), and Title 21 § 960 and 963 (conspiracy to import or export a controlled substance).
The biggest illicit cash crop in the United States is marijuana. Marijuana is typically used by drug smuggling organizations as a method to pay for other drugs, such as cocaine, ecstasy, heroin and N-benzylpiperazine (BZP). If law enforcement is able to disrupt the flow of marijuana, it is likely that the importation and street sales of other illicit drugs will decrease because marijuana proceeds are used to pay for other drugs. As a federal law enforcement officer, one of the methods that can be used to link suspects to a crime is through the comparison of seized drug shipments. One method of comparing seized shipments of Cannabis is through the utilization of DNA techniques, such as randomly amplified polymorphic DNAs (RAPDs), amplified fragment length polymorphisms (AFLPs) and short tandem repeats (STRs). These types of DNA comparisons can be instrumental in identifying growers, shippers, distributors and transporters, as well as identifying geographical locations where the drug was grown. By creating a marijuana DNA database, investigators and scientists will be able to produce a viable mechanism to link seized marijuana shipments to growers, distributors, transporters and to specific geographical locations.
There are several analytical techniques that can be utilized to aid law enforcement agents in identifying a drug in question. Upon seizing a shipment of an unknown drug (in this case, suspected marijuana), the samples are first entered into evidence via chain of custody. The samples are then taken to a forensic lab and are analyzed for identification purposes. After the identification process is complete, the second analytical step to be undertaken is individualization. This process incorporates DNA based methods as a technique to characterize individualization (also known as source attribution). Individualization can be used by AFLPs in order to construct future databases to determine if bulk shipments of seized marijuana share any common genetic profiles (Miller-Coyle et al. 2002). Furthermore, through AFLP analysis, the results may assist investigators in linking the shipment to the country of origin (Gough 1991).
The use of botanical evidence is not a unique concept in forensic science. Plant DNA has been used in the past to conduct both taxonomic and phylogenetic studies (Fanagan et al. 1994). Plant evidence has also been used successfully by investigators in solving murders, rapes and other crimes (Bock and Norris 1997), and most notably, in the 1993 Palo verde homicide case in Arizona (Yoon 1993). Suspected Cannabis can be identified through overall leaf structure, plant morphology and examination for the presence of microscopic structures called cystolith hairs (Nakamura 1969, Thornton and Nakamura 1972). Although cystolith hairs can aid the investigator in the identification of the plant, it is not conclusive because there are well over 80 plant species that have similar cystolith hair morphologies (Nakamura 1969). Gross morphology can be used to identify the genus and species of plants, but it can rarely show the origin of the plant (Miller-Coyle et al. 2005).
Biochemical techniques include gas chromatography (GC), high performance liquid chromatography (HPLC) (Gough 1991) and as a presumptive test, the Duquenois-Levine analysis (Butler 1962). Utilization of the Duquenois-Levine test can instantly offer the investigator with a rapid test that provides probable cause to believe the suspected drug is marijuana. Once a positive reaction is observed in the test kit, this will provide the investigator with probable cause to seek an arrest, search or seizure warrant on a suspected person or property. GC and HPLC aid the criminal investigator in identifying that the drug in question is marijuana through detection of its active ingredient, known as D9-tetrahydrocannabinol (THC)(Gillan et al. 1995, Grotenhermen 2003). GC or HPLC are routinely used for lab analysis to confirm the presence of THC, which the subsequent results would be introduced into court, as opposed to the presumptive Duquenois-Levine test (which typically is not used in court). GC and HPLC do not provide the investigator with information as to how particular seizures are linked; therefore, another method must be utilized to show linkages to a crime scene or to a person of interest.
There are three alternatives in localizing DNA from plant cells. DNA can be extracted from mitochondria, chloroplast or from the nucleus (which is the most common source of DNA in plants). One such method is through the utilization of the DNA, which can be extracted from seized marijuana plants through DNA analysis (Gillian et al. 1995). Recently, studies have been underway to track marijuana distribution patterns (Miller-Coyle et al. 2001, Miller-Coyle et al. 2003b). An alternative method that can be employed when identifying marijuana samples is through molecular genetics, such as by cloning and sequencing nuclear ribosomal (rDNA) in the internal transcribed spacer regions designated as ITS1 and ITS2 (Siniscalco and Caputo 1997). Investigators have not been able to determine the diversity of the genetic population of marijuana due to the illicitness of propagating plants covertly (Miller-Coyle et al. 2003). Some law enforcement crime labs have taken the first steps in surveying seized marijuana plants in an effort to assess the genetic diversity of the drug.
Upon identification of various marijuana samples, investigators often seek links between growers, transporters, and distributors. By linking the growers, transporters, and distributors, investigators can potentially prove conspiracy cases. Cannabis spp. can be propagated in two ways, by planting seeds or by cloning techniques (Clark 1981, Miller-Coyle et al. 2001, Miller-Coyle et al. 2002, Miller-Coyle et al. 2003a). When selecting marijuana seeds from a random population, Cannabis spp. would display their own unique genotypes, which can also be similarly observed in the human population. In cloned versions of Cannabis spp., however, the genotypes would be identical, much like identical twins (Miller-Coyle et al. 2001, Miller-Coyle et al. 2003a).
Individualization in a seized marijuana sample is integral to an investigation because it allows investigators to potentially link the evidence to the source. There are three tested methods for individualization: randomly amplified polymorphic DNAs (RAPDs), short tandem repeats (STRs) and AFLPs.
Randomly Amplified Polymorphic DNAs
RAPD testing typically shows a moderate discrimination power and is not used as much as AFLP due to the lack of reproducibility between laboratories (Jones et al. 1997). In a RAPD analysis, the investigator utilizes a random sequence of polymerase chain reaction (PCR) primers consisting of 10-15 base oligomers. The PCR primers bind to the PCR product based on sequence homology (Miller-Coyle et al. 2003). The PCR products are then subjected to electrophoresis on a 1% agarose gel, stained with ethidium bromide and ultimately interpreted through the observed banding patterns.
Short Tandem Repeats
STRs are based on short repeat bases, usually between 2-6 base pairs, that are repeated within nuclear DNA. When conducting STR analysis, specific PCR primers are used to bind to known sites within the nuclear DNA. Recently, 11 loci were screened in 40 samples of marijuana to accurately confirm reproducibility and accuracy, which showed 100% concordance (Alghanim and Almirall 2003). Since STR loci in marijuana has only been located recently, more time is required to validate future testing because mapping marijuana chromosomes has yet to be completed. Further testing is needed for locus independence, additional typing of core samples from a population is required to compare loci to discriminate between individual samples (individuals), estimates of inbreeding within the population needs to be examined and multiplexing of loci needs to be conducted in order to increase the power of discrimination (Miller-Coyle et al. 2003). Once Cannabis spp. STR loci are identified and validated, they will undoubtedly be used by investigators to establish links between sources and the samples on both a domestic and international level.
Amplified Fragment Length Polymorphisms
AFLPs have been widely used in several aspects of forensic sciences, such as distinguishing between individuals of the same species, which is inclusive of plants (Saunders et al. 2001). AFLP is of particular interest because it has the ability to distinguish between inbred genetic lines (Vos et al. 1995) and on single source biological evidence samples (Miller-Coyle et al. 2002, Miller-Coyle et al. 2003a). During an AFLP analysis, amplification of restriction fragments is detected with a DNA sequencer once the PCR primers recognize adaptor oligomers. The AFLP analysis method utilizes a laser that excites a fluorescent dye, which is integrated into the DNA fragments during PCR amplification. The fluorescent DNA fragments are processed through a camera as they pass by the laser, and the resulting band patterns are recorded by a computer. AFLPs are extremely useful in that they can be used to identify cloned Cannabis spp. generations, which can aid the investigator with identification of the source of supply. Analysis of cloned generations by AFLP has been proven to be highly reproducible. In unrelated Cannabis spp. samples, AFLP analysis was highly distinguishable (Miller-Coyle et al. 2003b).
AFLP has recently been validated for these purposes for the future introduction into a court setting. Recent experiments have demonstrated that regardless of what portion of the plant DNA is extracted (floral bud, stem, or a mature leaf), AFLP analysis showed identical genetic profiles, proving conclusively that AFLP typing is not dependant on the portion of the plant that has been examined (Miller-Coyle et al., 2002).
AFLP has also been validated when cloned marijuana plants were seized and analyzed from separate locations. Based on source information, an informant provided agents with two locations where marijuana was being grown. In one location, plants were being grown in pots under lights. In the second location, marijuana plants were being grown hydroponically. Both locations were raided, and the seized marijuana plants were subjected to AFLP. AFLP analysis showed that several subsets of the seized marijuana plants exhibited the same genetic profile. For the criminal investigator, this type of genetic evidence allows him/her to not only arrest the subject(s), but also increase the sentencing guidelines because more marijuana plants can be attributed to the same grow operation. It also shows that the two locations are linked, and the suspects are either sharing the same plants, or they were obtaining them from a common source (Miller-Coyle et al. 2002).
In cases where there are different marijuana samples to be analyzed, which is most likely the case when trying to compare two or more seized shipments from different locations, AFLP can be utilized to identify common profiles generated by different PCR primers. When AFLP profiles are analyzed, they can be scored based upon the presence or absence of peaks, thus assigning a binary code to them. For example, if a peak were present, it would be scored 1, when no peak is observed it would be assigned a value of 0. Each primer pair would be represented by a defined number of markers that could be scored and represented by the number of samples being tested. If 50 samples were being analyzed, then a total of 100 AFLP markers would be scored based upon two primer sets (Gillian et al. 1995). This type of analysis would be useful in determining if any of the samples showed similar profiles, which again could be used to link shipments, geographical locations, people or other crime scenes.
Identification of Cloned Marijuana Through DNA Tying
As the Assistant Special Agent in Charge for DHS-ICE on the northwest border of Washington, I routinely observe large amounts of marijuana, cocaine, heroin, ecstasy and BZP smuggled into and out of the United States from Canada and Mexico. A common trend in northwest Washington and Southwest Canada shows increased levels of marijuana harvested in covert indoor grow operations, thus producing large quantities of marijuana having a high THC content (personal communication, Sergeant Glen Anderson, RCMP). Propagation is routinely facilitated through cloning by taking cuttings from known high THC parent mother plants, then growing them in either soil or hydroponic grow operations (Miller-Coyle et al. 2001). Cloning is actually a form of asexual reproduction because there is a lack of meiotic recombination. As a result of cloning, subsequent plants carry identical genetic information from the mother plant. Cloning is very desirable because growers develop marijuana plants that exhibit desirable traits, such as high THC content.
Illicit drug cultivators thereby produce a large number of cloned plants that yield high concentrations of THC, thus commanding large sums of money per kilogram (dependant on the geographical location, upwards of $6,000-$10,000 per kilogram). DNA typing through utilization of AFLP (most common), RAPD or STR allows the criminal investigator to link grow operations to suppliers, distributors, and transporters. By having identical DNA profiles, a common source can be backtracked and identified. Once the origin is identified, agents can analyze distribution patterns of seized shipments based on identical DNA profiles or similar DNA profiles (in the event plants are propagated from seeds). As a simplistic example, a grower in Canada has propagated and sold 1,000 kilograms of high quality marijuana exhibiting a defined and specific DNA profile. Agents from San Diego, Los Angeles, Chicago and New York have seized 250 kilograms in each location and subsequently subjected samples from each shipment to AFLP analysis. All shipments showed the same DNA profile. The investigator designs a link chart showing relationships of the seized marijuana, which would show that the shipments had identical DNA profiles. The samples then can be linked back to where they were seized and from whom they were taken. By creating flow charts, the investigator can develop a picture of a drug smuggling and distribution network in order to track the distribution pattern back to a common point of origin, all from a few shipments of marijuana.
Often, distributors transport small quantities of marijuana from their residence "stash location" to prospective buyers. Routinely, the quantities are less than 100 pounds and it is difficult for federal agents to obtain authorization to prosecute individuals possessing or transporting smaller quantities of marijuana, especially in liberal districts such as the 9th circuit (Alaska, Washington, Oregon, Arizona, Idaho, Montana and California). Additionally, drug traffickers are so well schooled that they often claim that they did not have any knowledge that they were transporting marijuana, which in the 9th circuit is enough to have prosecution declined or dismissed. However, if agents can show that the 100-pound marijuana shipment has identical DNA profiles to another shipment that the defendant was previously linked, the probability of a prosecution is significantly increased because the agent has now shown a definitive link from one shipment to another shipment.
AFLP can also be used on the state level when attempting to link street level dealers to distributors and users. As in the federal system, distributors and organizers receive much more severe penalties for distribution as compared to personal consumption. In order to show a suspect is a supplier or distributor, the investigator can utilize several smaller shipments of marijuana that were seized at different locations to link it to a common source (Gillan et al. 1995). If local police conduct "buy walk operations" and purchase one-ounce samples from 10 different people from 10 different locations, and AFLP shows identical DNA profiles, the investigator can conclude that the source of supply is probably from the same primary distributor.
Propagation of marijuana by seeds
Marijuana is a dioecious plant that when propagated through seeds are facilitated through sexual reproduction between a male (staminate) and female (pistillate) flowers. Once there is pollination, new plants are produced with a unique recombinant genotype. In contrast to cuttings (cloned plants), marijuana that has been raised from a seed will exhibit a unique DNA profile, just as each human or other individual within a species (excluding identical twins). By subjecting Cannabis spp. to DNA analysis that were grown from seeds, it would provide the investigator with an armament of molecular evidence to link a marijuana leaf to an individual, vehicle, or property. Through personal experience, it is necessary to be able to show that a false compartment was used to smuggle marijuana into the United States from Canada. False compartments alone are not enough to prosecute an individual for drug smuggling because agents must prove beyond a reasonable doubt that drugs were concealed in the compartment. If agents are able to show that there was any residue left behind in the compartment, and it contained detectable amounts of drugs, not only can the vehicle be seized, but the driver/occupant can be arrested. There are times when residue has been dried and standard GC or HPLC techniques are not sufficient to conduct a viable test. In these types of situations, AFLP could be used to obtain the genetic profile of the substance. If there was a marijuana DNA database, results could then be compared to other profiles to determine if it had any similar profiles to other seized shipments. This would accomplish two things for the investigator, (1) it would be sufficient proof needed to seize the vehicle, and (2) it could link the suspect to another illegal marijuana shipment if he/she had been arrested before. If the defendant had two prior felony convictions, he/she could be a third striker, which carries severe penalties and increased amounts of jail time.
Although millions of pounds of marijuana are seized by law enforcement each year, surprisingly there are very few databases to compare seized samples against one another. Several factors need to be considered when constructing a genomic DNA marijuana database, such as the genetic diversity of plants originating from a wide variety of geographic locations. If the genetic diversity of marijuana is contemplated on an evolutionary scale, sampling must be done worldwide; however, if a database is to be constructed for the sole purposes of identifying and linking seized marijuana shipments to a defined geographical area (such as Canada, Mexico and the United States), sampling can be done on a smaller scale.
Many pieces of evidence are utilized in the criminal prosecution of a defendant. If using DNA obtained from marijuana to show links between a seized sample and the defendant, individualization of the sample is essential to establish a link. There are many other potential uses for individualization via RAPD, AFLP or STR in future forensic analysis of marijuana. Such forensic links might assist the investigator in connecting a potential defendant to rapes, homicides and other crimes of violence when marijuana evidence is discovered at a crime scene. By analyzing the DNA contained in the marijuana, investigators might be able to establish its origin and backtrack it to a known grower or distributor, thus providing a potential suspect to a crime. Additionally, in the field of drug enforcement, agents might be able to locate specific geographic regions where Cannabis spp. is being grown and cultivated. In the case of hydroponic grow operations and cloned plants, individualization might be able to guide investigators to the location of growers, distributors and transporters through common DNA profiles that are obtained from seized shipments (Miller-Coyle et al. 2001, Miller-Coyle et al. 2002, Miller-Coyle et al. 2003b).
As law enforcement focuses in on outdoor grow operations in climates conducive to propagating marijuana through seedlings, more covert means are undertaken by growers to conceal where the illicit crops are being cultivated. Since law enforcement monitors internet sites where seeds can be purchased, growers are being more careful as to not attract attention to their illegal activities and are resulting in cloning high yield THC mother plants. Many drug dealers involved with the cultivation and subsequent harvesting of Cannabis spp. choose to clone their best producing plants because of concealment, having a controlled growing environment and relative safety from law enforcement. Throughout Washington and Canada, several organized groups utilize indoor grows and propagate Cannabis spp. through cloning techniques (Alghanim and Almirall 2003). Because more and more plants are being grown through cloned techniques, law enforcement has an opportunity to link major marijuana growers to smaller growers. As a result, law enforcement can establish distribution patterns and points of origin of seized marijuana shipments during search warrants and traffic stops. If in the event marijuana distributors are obtaining their product from seedlings, forensic applications can still be correlated to plant evidence that is discovered at a crime scene, on or near a suspect or a property.
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