This is on the DEA’s website. It’s an overview by the DEA of DNA Methods for the Identification and Individualization of Marjuana.
ABSTRACT: The purpose of this review is to summarize the status of DNA-based methods for the identification and individualization of marijuana. In forensics, both identification of a substance as marijuana and the subsequent individualization of a sample may be desired for casework. Marijuana identification methods in the United States primarily include biochemical tests and, less frequently, DNA-based tests. Under special circumstances, DNA-based tests can be useful. For example, if the quantity of seized marijuana is extremely small and/or biochemical tests do not detect any 9-tetrahydrocannabinol (THC), DNA identification of plant material as Cannabis is still possible. This circumstance can arise when seeds, trace residue, tiny leaf fragments, or fine roots need to be analyzed. Methods for the individualization of marijuana include Amplified Fragment Length Polymorphism (AFLP), Random Amplified Polymorphic DNA (RAPD), and Short Tandem Repeat (STR) techniques that link an evidentiary sample to a source. Marijuana growers propagate their plants either by seed or by cloning. Seed-generated marijuana plants are expected to have unique DNA profiles analogous to a human population. Cloned marijuana plants, however, exhibit identical DNA profiles that allow for tracking of plant material derived from a common genetic lineage. The authors have validated the AFLP method for marijuana samples and are constructing a comparative database of marijuana seizure samples to estimate the expected frequency of a DNA profile match between unrelated plants. Continued development of DNA-based methods for plants can be useful for marijuana and other types of plant evidence in forensics.
KEYWORDS: Cannabis; Polymorphism (Genetics); Polymorphism; Restriction Fragment Length; Random Amplified Polymorphic DNA Technique; Tandem Repeat Sequences
Cannabis sativa (marijuana) has existed since ancient times and is widely used as a fiber source, a food source, a medicine and a euphoriant (1-4). Marijuana has been used to treat a variety of ailments including glaucoma, pain, nausea, asthma, depression, neuralgia and insomnia (5). Like many crops (e.g. wheat, corn), marijuana was originally a naturally occurring weed species. It was bred and cultivated into a significant cash crop for a multi-billion dollar illicit industry. The hallucinogenic properties of marijuana are derived from ? 9-tetrahydrocannabinol (THC) (6) and potent (high-THC level) marijuana cultivars are sought by the discerning marijuana users (7,8). Before the mid-1970’s, the majority of U.S. marijuana was imported from Mexico. When the U.S. and Mexico co-operated in marijuana eradication programs, a domestic growing industry began in the United States. Most of the highly prized United States cultivars originated from a few breeding stocks from the West Coast (5). Now, seeds for marijuana growers are accessible worldwide via the Internet; seed catalogues are posted with extensive descriptions and price lists (e.g. www.cannabisseeds.org). The extent of genetic variation in marijuana populations is unknown due to the illicit nature of this substance and because growers propagate plants secretively. A survey of marijuana seizure samples using DNA profiling methods could be used to assess levels of genetic diversity within this crop.
There are two main steps in most forensic classification schemes that can be applied to marijuana seizure samples. The first step involves identification of a sample. For marijuana, both biochemical (9,10) and DNA tests (11-12) are available to identify a substance as Cannabis. The second step is individualization (source attribution). For marijuana, several DNA-based methods are under development and will be described in later sections. Biochemical methods to establish geographic origin of a plant have met with variable success (13-17). However, contaminants (18) and packaging (17) have shown a correlation with marijuana source. Biochemical profiling has also successfully differentiated between resinous and textile Cannabis (19), drug subgroups (marijuana, sinsemilla, Thai sticks, ditchweed) (7) and plant gender (20).
Cannabis can be seed-propagated or perpetuated through cloning (1,21,22). Seed-propagated plants are expected to have their own unique genotypes analogous to humans selected from a random population. However, plants that have been propagated through cloning should have identical genotypes like identical twins (21,22). Tracking cloned marijuana based on DNA should be relatively simple; seizure samples with identical profiles should have a common genetic source. The ability to link marijuana growers and users to a common distributor by DNA would be a useful investigative tool for narcotics enforcement. In addition, some forensic cases may be able to link a suspect and victim by matching marijuana samples. The Connecticut State Forensic Science Laboratory, along with several other research groups, is in the process of developing DNA-based methods for the individualization of plant (especially marijuana) samples that are seized from crime scenes. Different DNA-based techniques have different applications, benefits and limitations but all can be utilized to supplement existing forensic methods.
Marijuana Drug Facts
United States teenagers use marijuana more than any other drug according to the U. S. Government Substance Abuse and Mental Health Services Administration (23,24). For example, 20% of teenagers aged 12 to 17 years have used marijuana at least once (23,24). In comparison, only 3% of teenagers have used Ecstasy and approximately 2% report using cocaine (23,24). Marijuana prices vary depending on the quantity and quality of what is sold and where the consumer is geographically located, however; it is estimated that marijuana is a multi-billion dollar industry in the United States. One primary source of marijuana is from Mexico where the Border Patrol and U. S. Customs Service seize tons of marijuana worth millions of dollars every year (24). In addition to imported marijuana, the U.S. has a very profitable domestic marijuana growing industry (1,5,24,25).
According to the 2001 National Forensic Laboratory Information System (NFLIS) report, 36% of the analyzed drug items at the national level were identified as Cannabis compared to 33% as cocaine, 11% as methamphetamine and 8% as heroin, respectively (26). Considerable variation exists in drug types reported across different regions of the United States; it should be noted that these differences could result from different law enforcement strategies or laboratory analysis policies. In general, Cannabis is identified in 25% or more of the drug seizures for the United States regardless of geographical region. In 2001, Cannabis estimates for the Midwest, the Northeast, the South and the West were 47%, 36%, 36% and 23%, respectively (26).
In 1977, regional narcotics enforcement squads were replaced by a Statewide Narcotics Task Force in Connecticut (27). The Task Force is authorized to enforce the state laws concerning the manufacture, distribution, sale and possession of narcotics and controlled substances. In addition to enforcement, the Connecticut Statewide Narcotics Task Force collects and provides information regarding drug seizures for Connecticut on an annual basis (25,27). Both indoor and outdoor marijuana grow operations have been identified in Connecticut (25,27). The outdoor grow season in Connecticut begins in April and continues until harvest time in mid-September.
The indoor grow season is year-round. The Statewide Narcotics Task Force, in conjunction with the Drug Enforcement Administration (DEA), sponsors and coordinates the Domestic Cannabis Eradication/Suppression Program in Connecticut (25,27). For the first nine months of 2002, statistics for the Statewide Narcotics Task Force domestic Cannabis eradication program indicated while greater numbers of outdoor plots were identified compared to indoor grow operations, the number of plants seized were comparable between indoor and outdoor cultivation plots (Table 1) (27). Indoor grow operations may be increasing in number and scale or are more easily detected based on a comparison of data from the years 1999-2002 (Table 1) (27). According to Connecticut statistics for 2002, marijuana distribution and consumption has steadily increased and the demand for high quality hydroponically grown marijuana has also increased despite the greater cost to the consumer (25,27). Marijuana cultivated in Connecticut represents a small fraction of the amount consumed by its state residents. The majority of consumed marijuana is imported from California, Texas and Mexico (25,27)
Although Connecticut is a relatively small marijuana producing state, marijuana usage still continues to be a substantial drug problem. Based on statistics from the Connecticut Department of Public Safety Controlled Substances and Toxicology Laboratory, the percentage of reported marijuana for the past three years (2000-2002) has remained stable (approximately 27%) (28). Reported marijuana drug items are only exceeded by cocaine which averages 35% of the total drug items reported (28). The majority of Cannabis items reported by the Laboratory for 2000-2002 are from four of nine Connecticut counties (Waterbury, New Haven, Hartford and Fairfield) (28). However, marijuana drug items have been identified and seized from all areas of Connecticut (28). The majority of analyzed drug items reported by the Laboratory are comprised of a single identifiable drug substance; less than 1.5% of drug items were reported as drug mixtures (28).
Identifying a plant sample as Cannabis sativa is the first step in determining if an illegal substance has been seized. Methods for the identification of marijuana include: Botanical identification through inspection of the intact plant morphology and growth habit (1,2), microscopical examination of leaves for the presence of cystolith hairs (29-31), chemical screening tests such as the Duquenois-Levine test (32-34), THC identification through biochemical methods (10,19,33,35,36), and the use of molecular sequencing to identify DNA sequence homology to reference marijuana samples (11,12).
Biochemical testing is the most common method for identifying plant material as marijuana. Chemical tests include those developed by Duquenois and other modifications of the original Duquenois test (32-34). Other chemical tests are the Rutgers Identification for Marijuana (RIM) technique and use of gas liquid chromatography (GLC) and high-pressure liquid chromatography (HPLC) to identify cannabinoid compounds (10,15-17,19,35,36). Occasionally, some marijuana samples can’t be identified through chemical means because little or no THC is present. Such situations include seizures of seeds not associated with marijuana plant leaves and cases where the plants have been harvested but the roots have been left at the crime scene. In these situations, DNA testing can provide a means for marijuana identification that would otherwise not be possible.
Although three forms of DNA are present in plant cells (mitochondrial, chloroplast and nuclear), nuclear DNA sequences are most commonly used for plant species identification. DNA-based tests for the identification of marijuana include the molecular analysis of the ITS1, ITS2 and trnL intron (11,12,63,64). A comparison of the ITS1 and ITS2 Polymerase Chain Reaction (PCR) product sizes in five samples of marijuana and in one sample of a close relative (Humulus lupulus) revealed a size difference between marijuana and Humulus for the ITS2 region (11,12). Other tests using PCR amplification and subsequent restriction enzyme digestion of the trnL region of the chloroplast has shown that marijuana DNA profiles can be generated and compared between samples and may be useful for forensic purposes (11,12).