Until late, forensic vegetation has been an under utilised resource for associating seized works grounds to felons, victims and offense scenes. Forensic vegetation has non been used much chiefly because of deficiency of cognition among offense scene research workers, constabulary and prosecuting officers ( Bock and Norris 1997 ) .
Botanic grounds can be instrumental in a broad assortment of probes, including homicides, snatchs, colzas, and drug smuggling and distribution operations.Drug smuggling and trafficking are a serious job in the United States. In 2008 entirely, the Department of Homeland Security ( DHS ) and the Drug Enforcement Administration ( DEA ) seized a combined 1,144,374 kgs of marihuana, 103,795 kgs of cocaine and 1,653 kgs of diacetylmorphine ( personal communicating, Matthew Stentz, DHS-ICE, USDOJ 2009 ) . These figures merely represent the tip of the iceberg, and do non include other federal, province or local bureau ictuss, or drugs that were non detected and later seized by agents.Significant measures of marihuana ( chiefly Cannabis sativa ) are smuggled into the United States from Mexico and Canada. One of the primary directives of the DHS and DEA is to do every effort to place, interrupt and level drug smuggling organisations. Once these organisations are identified, it is up to the condemnable research worker to develop sufficient likely cause to collar and prosecute persons that are associated with this illicit trade. Agents and research workers are besides responsible for associating distributers, providers and transporters to turn out drug smuggling/trafficking confederacies.
Once agents obtain plenty likely cause to collar suspects and hunt and prehend grounds and belongingss, the suspects are later charged with misdemeanors of United States jurisprudence, to wit, Title 18 § 371 ( confederacy ) , Title 21 § 841 ( a ) ( 1 ) and Title 21 § 846 ( confederacy to possess with purpose to administer a controlled substance ) , and Title 21 § 960 and 963 ( confederacy to import or export a controlled substance ) .The biggest illicit hard currency harvest in the United States is marijuana. Marijuana is typically used by drug smuggling organisations as a method to pay for other drugs, such as cocaine, rapture, diacetylmorphine and N-benzylpiperazine ( BZP ) .
If jurisprudence enforcement is able to interrupt the flow of marihuana, it is likely that the importing and street gross revenues of other illicit drugs will diminish because marijuana returns are used to pay for other drugs. As a federal jurisprudence enforcement officer, one of the methods that can be used to associate suspects to a offense is through the comparing of seized drug cargos. One method of comparing seized cargos of Cannabis is through the use of DNA techniques, such as indiscriminately amplified polymorphous DNAs ( RAPDs ) , amplified fragment length polymorphisms ( AFLPs ) and short tandem repetitions ( STRs ) .
These types of DNA comparings can be instrumental in placing agriculturists, shippers, distributers and transporters, every bit good as placing geographical locations where the drug was grown. By making a marihuana DNA database, research workers and scientists will be able to bring forth a feasible mechanism to associate seized marihuana cargos to agriculturists, distributers, transporters and to specific geographical locations.
There are several analytical techniques that can be utilized to help jurisprudence enforcement agents in placing a drug in inquiry. Upon prehending a cargo of an unknown drug ( in this instance, suspected marihuana ) , the samples are first entered into grounds via concatenation of detention.
The samples are so taken to a forensic lab and are analyzed for designation intents. After the designation procedure is complete, the 2nd analytical measure to be undertaken is individualisation. This procedure incorporates DNA based methods as a technique to qualify individualisation ( besides known as beginning ascription ) . Individualization can be used by AFLPs in order to build future databases to find if bulk cargos of seized marihuanas portion any common familial profiles ( Miller-Coyle et Al.
2002 ) . Furthermore, through AFLP analysis, the consequences may help research workers in associating the cargo to the state of beginning ( Gough 1991 ) .
The usage of botanical grounds is non a alone construct in forensic scientific discipline. Plant DNA has been used in the yesteryear to carry on both systematic and phyletic surveies ( Fanagan et al. 1994 ) . Plant grounds has besides been used successfully by research workers in work outing slayings, colzas and other offenses ( Bock and Norris 1997 ) , and most notably, in the 1993 Palo verde homicide instance in Arizona ( Yoon 1993 ) . Suspected Cannabis can be identified through overall foliage construction, works morphology and scrutiny for the presence of microscopic constructions called bladder stone hairs ( Nakamura 1969, Thornton and Nakamura 1972 ) .
Although cystolith hairs can help the research worker in the designation of the works, it is non conclusive because there are good over 80 works species that have similar bladder stones hair morphologies ( Nakamura 1969 ) . Gross morphology can be used to place the genus and species of workss, but it can seldom demo the beginning of the works ( Miller-Coyle et Al. 2005 ) .
Biochemical techniques include gas chromatography ( GC ) , high public presentation liquid chromatography ( HPLC ) ( Gough 1991 ) and as a presumptive trial, the Duquenois-Levine analysis ( Butler 1962 ) .
Use of the Duquenois-Levine trial can immediately offer the research worker with a rapid trial that provides likely cause to believe the suspected drug is marijuana. Once a positive reaction is observed in the trial kit, this will supply the research worker with likely cause to seek an apprehension, hunt or ictus warrant on a suspected individual or belongings. GC and HPLC aid the condemnable research worker in placing that the drug in inquiry is marijuana through sensing 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 corroborate the presence of THC, which the subsequent consequences would be introduced into tribunal, as opposed to the presumptive Duquenois-Levine trial ( which typically is non used in tribunal ) . GC and HPLC do non supply the research worker with information as to how peculiar ictuss are linked ; hence, another method must be utilized to demo linkages to a offense scene or to a individual of involvement.
Deoxyribonucleic acid Localization
There are three options in placing Deoxyribonucleic acid from works cells. Deoxyribonucleic acid can be extracted from chondriosome, chloroplast or from the karyon ( which is the most common beginning of DNA in workss ) . One such method is through the use of the DNA, which can be extracted from seized marihuana workss through DNA analysis ( Gillian et Al. 1995 ) . Recently, surveies have been afoot to track marijuana distribution forms ( Miller-Coyle et Al. 2001, Miller-Coyle et Al.
2003b ) . An alternate method that can be employed when placing marihuana samples is through molecular genetic sciences, such as by cloning and sequencing atomic ribosomal ( rDNA ) in the internal transcribed spacer parts designated as ITS1 and ITS2 ( Siniscalco and Caputo 1997 ) . Research workers have non been able to find the diverseness of the familial population of marihuanas due to the illicitness of propagating workss covertly ( Miller-Coyle et Al. 2003 ) . Some jurisprudence enforcement offense labs have taken the first stairss in appraising seized marihuana workss in an attempt to measure the familial diverseness of the drug.
Upon designation of assorted marihuana samples, research workers frequently seek links between agriculturists, transporters, and distributers. By associating the agriculturists, transporters, and distributers, research workers can potentially turn out confederacy instances.
Cannabis spp. can be propagated in two ways, by seting seeds or by cloning techniques ( Clark 1981, Miller-Coyle et Al. 2001, Miller-Coyle et Al. 2002, Miller-Coyle et Al. 2003a ) .
When choosing marihuanas seeds from a random population, Cannabis spp. would expose their ain alone genotypes, which can besides be likewise observed in the human population. In cloned versions of Cannabis spp. , nevertheless, the genotypes would be indistinguishable, much like indistinguishable twins ( Miller-Coyle et Al. 2001, Miller-Coyle et Al. 2003a ) .Individualization in a seized marihuana sample is built-in to an probe because it allows research workers to potentially associate the grounds to the beginning.
There are three tried methods for individualisation: indiscriminately amplified polymorphous DNAs ( RAPDs ) , short tandem repetitions ( STRs ) and AFLPs.
Randomly Amplified Polymorphic DNAs
RAPD proving typically shows a moderate favoritism power and is non used every bit much as AFLP due to the deficiency of duplicability between research labs ( Jones et al. 1997 ) . In a RAPD analysis, the research worker utilizes a random sequence of polymerase concatenation reaction ( PCR ) primers dwelling of 10-15 base oligomers. The PCR primers bind to the PCR merchandise based on sequence homology ( Miller-Coyle et Al. 2003 ) . The PCR merchandises are so subjected to electrophoresis on a 1 % agarose gel, stained with ethidium bromide and finally interpreted through the ascertained stria forms.
Short Tandem Repeats
STRs are based on short repetition bases, normally between 2-6 base brace, that are repeated within atomic DNA.
When carry oning STR analysis, specific PCR primers are used to adhere to cognize sites within the atomic DNA. Recently, 11 venue were screened in 40 samples of marihuana to accurately corroborate duplicability and truth, which showed 100 % harmony ( Alghanim and Almirall 2003 ) . Since STR venue in marihuana has merely been located late, more clip is required to formalize future testing because mapping marihuana chromosomes has yet to be completed. Further testing is needed for venue independency, extra typewriting of nucleus samples from a population is required to compare venue to know apart between single samples ( persons ) , estimations of inbreeding within the population needs to be examined and multiplexing of venue demands to be conducted in order to increase the power of favoritism ( Miller-Coyle et Al. 2003 ) . Once Cannabis spp. STR venue are identified and validated, they will doubtless be used by research workers to set up links between beginnings and the samples on both a domestic and international degree.
Amplified Fragment Length Polymorphisms
AFLPs have been widely used in several facets of forensic scientific disciplines, such as separating between persons of the same species, which is inclusive of workss ( Saunders et al. 2001 ) . AFLP is of peculiar involvement because it has the ability to separate between inbred familial lines ( Vos et al. 1995 ) and on individual beginning biological grounds samples ( Miller-Coyle et Al. 2002, Miller-Coyle et Al.
2003a ) . During an AFLP analysis, elaboration of limitation fragments is detected with a Deoxyribonucleic acid sequenator once the PCR primers recognize adapter oligomers. The AFLP analysis method utilizes a optical maser that excites a fluorescent dye, which is integrated into the Deoxyribonucleic acid fragments during PCR elaboration. The fluorescent Deoxyribonucleic acid fragments are processed through a camera as they pass by the optical maser, and the ensuing set forms are recorded by a computing machine. AFLPs are highly utile in that they can be used to place cloned Cannabis spp. coevalss, which can help the research worker with designation of the beginning of supply. Analysis of cloned coevalss by AFLP has been proven to be extremely consistent. In unrelated Cannabis spp.
samples, AFLP analysis was extremely distinguishable ( Miller-Coyle et Al. 2003b ) .AFLP has late been validated for these intents for the future debut into a tribunal scene. Recent experiments have demonstrated that regardless of what part of the works DNA is extracted ( flowered bud, root, or a mature foliage ) , AFLP analysis showed indistinguishable familial profiles, turn outing once and for all that AFLP typewriting is non dependent on the part of the works that has been examined ( Miller-Coyle et al. , 2002 ) .AFLP has besides been validated when cloned marihuana workss were seized and analyzed from separate locations.
Based on beginning information, an source provided agents with two locations where marihuana was being grown. In one location, workss were being grown in pots under visible radiations. In the 2nd location, marihuana workss were being grown hydroponically. Both locations were raided, and the seized marihuana workss were subjected to AFLP. AFLP analysis showed that several subsets of the seized marihuana workss exhibited the same familial profile.
For the condemnable research worker, this type of familial grounds allows him/her to non merely collar the topic ( s ) , but besides increase the condemning guidelines because more marijuana workss can be attributed to the same grow operation. It besides shows that the two locations are linked, and the suspects are either sharing the same workss, or they were obtaining them from a common beginning ( Miller-Coyle et Al. 2002 ) .In instances where there are different marihuanas samples to be analyzed, which is most likely the instance when seeking to compare two or more seized cargos from different locations, AFLP can be utilized to place common profiles generated by different PCR primers. When AFLP profiles are analyzed, they can be scored based upon the presence or absence of extremums, therefore delegating a binary codification to them.
For illustration, if a extremum were present, it would be scored 1, when no extremum is observed it would be assigned a value of 0. Each primer brace would be represented by a defined figure of markers that could be scored and represented by the figure of samples being tested. If 50 samples were being analyzed, so a sum of 100 AFLP markers would be scored based upon two primer sets ( Gillian et Al. 1995 ) . This type of analysis would be utile in finding if any of the samples showed similar profiles, which once more could be used to associate cargos, geographical locations, people or other offense scenes.
Designation of Cloned Marijuana Through DNA Tying
As the Assistant Special Agent in Charge for DHS-ICE on the northwest boundary line of Washington, I routinely observe big sums of marihuana, cocaine, diacetylmorphine, ecstasy and BZP smuggled into and out of the United States from Canada and Mexico.
A common tendency in northwest Washington and Southwest Canada shows increased degrees of marihuana harvested in covert indoor grow operations, therefore bring forthing big measures of marihuana holding a high THC content ( personal communicating, Sergeant Glen Anderson, RCMP ) . Propagation is routinely facilitated through cloning by taking film editings from known high THC parent female parent workss, so turning them in either dirt or aquicultural grow operations ( Miller-Coyle et Al. 2001 ) .
Cloning is really a signifier of nonsexual reproduction because there is a deficiency of meiotic recombination. As a consequence of cloning, subsequent workss carry indistinguishable familial information from the female parent works. Cloning is really desirable because agriculturists develop marijuana workss that exhibit desirable traits, such as high THC content.
Illicit drug agriculturists thereby produce a big figure of cloned workss that yield high concentrations of THC, therefore commanding big amounts of money per kg ( dependent on the geographical location, upwards of $ 6,000- $ 10,000 per kg ) . Deoxyribonucleic acid typing through use of AFLP ( most common ) , RAPD or STR allows the condemnable research worker to associate turn operations to providers, distributers, and transporters. By holding indistinguishable DNA profiles, a common beginning can be backtracked and identified.
Once the beginning is identified, agents can analyse distribution forms of seized cargos based on indistinguishable DNA profiles or similar Deoxyribonucleic acid profiles ( in the event workss are propagated from seeds ) . As a simplistic illustration, a agriculturist in Canada has propagated and sold 1,000 kgs of high quality marijuana exhibiting a defined and specific DNA profile. Agents from San Diego, Los Angeles, Chicago and New York have seized 250 kgs in each location and later subjected samples from each cargo to AFLP analysis. All cargos showed the same DNA profile. The research worker designs a link chart demoing relationships of the seized marihuana, which would demo that the cargos had indistinguishable DNA profiles. The samples so can be linked back to where they were seized and from whom they were taken. By making flow charts, the research worker can develop a image of a drug smuggling and distribution web in order to track the distribution form back to a common point of beginning, all from a few cargos of marihuana.
Frequently, distributers transport little measures of marihuana from their abode “ stash location ” to prospective purchasers. Routinely, the measures are less than 100 lbs and it is hard for federal agents to obtain mandate to prosecute persons possessing or transporting smaller measures of marihuana, particularly in broad territories such as the 9th circuit ( Alaska, Washington, Oregon, Arizona, Idaho, Montana and California ) . Additionally, drug sellers are so good schooled that they frequently claim that they did non hold any cognition that they were transporting marihuana, which in the 9th circuit is adequate to hold prosecution declined or dismissed. However, if agents can demo that the 100-pound marihuana cargo has indistinguishable DNA profiles to another cargo that the suspect was antecedently linked, the chance of a prosecution is significantly increased because the agent has now shown a unequivocal nexus from one cargo to another cargo.AFLP can besides be used on the province degree when trying to associate street degree traders to distributers and users. As in the federal system, distributers and organisers receive much more terrible punishments for distribution as compared to personal ingestion. In order to demo a suspect is a provider or distributer, the research worker can use several smaller cargos of marihuana that were seized at different locations to associate it to a common beginning ( Gillan et al. 1995 ) .
If local constabulary behavior “ purchase walk operations ” and buy one-ounce samples from 10 different people from 10 different locations, and AFLP shows indistinguishable DNA profiles, the research worker can reason that the beginning of supply is likely from the same primary distributer.
Propagation of marihuana by seeds
Marijuana is a dioecian works that when propagated through seeds are facilitated through sexual reproduction between a male ( staminate ) and female ( pistillate ) flowers. Once there is pollenation, new workss are produced with a alone recombinant genotype. In contrast to film editings ( cloned workss ) , marihuana that has been raised from a seed will exhibit a alone DNA profile, merely as each homo or other single within a species ( excepting indistinguishable twins ) . By subjecting Cannabis spp. to DNA analysis that were grown from seeds, it would supply the research worker with an armament of molecular grounds to associate a marihuana foliage to an person, vehicle, or belongings. Through personal experience, it is necessary to be able to demo that a false compartment was used to smuggle marihuana into the United States from Canada. False compartments entirely are non plenty to prosecute an person for drug smuggling because agents must turn out beyond a sensible uncertainty that drugs were concealed in the compartment.
If agents are able to demo that there was any residue left buttocks in the compartment, and it contained noticeable sums of drugs, non merely 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 non sufficient to carry on a feasible trial. In these types of state of affairss, AFLP could be used to obtain the familial profile of the substance. If there was a marijuana DNA database, consequences could so be compared to other profiles to find if it had any similar profiles to other seized cargos. This would carry through two things for the research worker, ( 1 ) it would be sufficient cogent evidence needed to prehend the vehicle, and ( 2 ) it could associate the suspect to another illegal marihuana cargo if he/she had been arrested before. If the suspect had two anterior felony strong beliefs, he/she could be a 3rd striker, which carries terrible punishments and increased sums of gaol clip.
Although 1000000s of lbs of marihuana are seized by jurisprudence enforcement each twelvemonth, surprisingly there are really few databases to compare seized samples against one another. Several factors need to be considered when building a genomic DNA marihuana database, such as the familial diverseness of workss arising from a broad assortment of geographic locations.
If the familial diverseness of marihuana is contemplated on an evolutionary graduated table, trying must be done worldwide ; nevertheless, if a database is to be constructed for the exclusive intents of identifying and associating seized marihuana cargos to a defined geographical country ( such as Canada, Mexico and the United States ) , trying can be done on a smaller graduated table.Many pieces of grounds are utilized in the condemnable prosecution of a suspect. If utilizing DNA obtained from marihuana to demo links between a seized sample and the suspect, individualisation of the sample is indispensable to set up a nexus.
There are many other possible utilizations for individualisation via RAPD, AFLP or STR in future forensic analysis of marihuana. Such forensic links might help the research worker in linking a possible suspect to ravish, homicides and other offenses of force when marihuana grounds is discovered at a offense scene. By analysing the Deoxyribonucleic acid contained in the marihuana, research workers might be able to set up its beginning and turn back it to a known agriculturist or distributer, therefore supplying a possible suspect to a offense. Additionally, in the field of drug enforcement, agents might be able to turn up specific geographic parts where Cannabis spp. is being grown and cultivated. In the instance of aquicultural grow operations and cloned workss, individualisation might be able to steer research workers to the location of agriculturists, distributers and transporters through common DNA profiles that are obtained from seized cargos ( Miller-Coyle et Al.
2001, Miller-Coyle et Al. 2002, Miller-Coyle et Al. 2003b ) .
As jurisprudence enforcement focuses in on out-of-door grow operations in climes conducive to propagating marihuanas through seedlings, more covert agencies are undertaken by agriculturists to hide where the illicit harvests are being cultivated. Since jurisprudence enforcement proctors internet sites where seeds can be purchased, agriculturists are being more careful as to non pull attending to their illegal activities and are ensuing in cloning high output THC female parent workss. Many drug traders involved with the cultivation and subsequent harvest home of Cannabis spp. take to clone their best bring forthing workss because of privacy, holding a controlled growth environment and comparative safety from jurisprudence 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 workss are being grown through cloned techniques, jurisprudence enforcement has an chance to associate major marihuana agriculturists to smaller agriculturists. As a consequence, jurisprudence enforcement can set up distribution forms and points of beginning of seized marihuana cargos during search warrants and traffic Michigans. If in the event marihuana distributers are obtaining their merchandise from seedlings, forensic applications can still be correlated to works grounds that is discovered at a offense scene, on or near a suspect or a belongings.
- Alghanim, H. J. and J.
R. Almirall. 2003.
Development of microsatellite markers in Cannabis sativa for DNA typewriting and familial relatedness analyses. Anal Bioanal Chem. 376 ( 8 ) : 1225-1233.
- Anderson, G.
2009. Personal communicating.
- Bock, J. H. and D.
O. Norris. 1997. Forensic vegetation: An under-utilized resource. J Forensic Sci. 42: 364-367.
- Butler, W. Duquenois-Levine trial for marihuana. 1962. J Assoc Off Anal Chem. 45: 597-600.
- Clark, R.
C. Marijuana vegetation: An advanced survey: The extension and genteelness of typical Cannabis. 1981. Ronin Publishing, Berkley, CA.
- Gillian R. , M. Cole, A. Linacre, J.
Thorpe and, N. Watson. 1995. Comparison of Cannabis sativa by random elaboration of polymorphous DNA ( RAPD ) and HPLC of cannabinoids: a preliminary survey.
Science and Justice. 35: 169-177.
- Gough, T. A.
1991. Examination of drugs in smuggling discourtesies, pp. 511-567. In T. A. Gough ( ed. ) , The analysis of drugs of maltreatment. J Wiley and Sons, Chichester, United Kingdom.
- Grotenhermen, F. 2003. Pharmacokinetics and pharmacodynamics of cannabinoids. Clin Pharmacokinet. 42: 327-60.
- Jones, C. J. , K.
J. Edwards, S. Castaglione, M. O.
Winfield, F. Sala and, C. van de Wiel. 1997. Reproducibility testing of RAPD, AFLP and SSR markers in workss by a web of European research labs. Mol Breed. 3:381-390
- Miller-Coyle, H. , C.
Ladd, T. Palmbach, and, H. Lee. 2001. The green revolution: Contributions to forensics and drug enforcement.
Croat Med J. 42 ( 3 ) : 340-345
- Miller-Coyle H. , J. Germano-Presby, C. Ladd, T.
Palmbach and, H. C. Lee. Tracking clonal marihuana utilizing amplified fragment length polymorphism ( AFLP ) analysis: an overview. Proceedings of the 13th International Symposium on Human Identification, 7-13 October 2002, Phoenix, AZ.
- Miller-Coyle, H. , T. Palmbach, N.
Juliano, C. Ladd and, H. Lee. 2003a.
An Overview of DNA Methods for the Identification and Individualization of Marijuana. Croat Med J. 44 ( 3 ) : 315-321.
- Miller-Coyle H. , G. Shutler, S.
Abrams, J. Hanniman, S. Neylon, C. Ladd, T.
Palmback and H. C. Lee. 2003b.
A simple DNA extraction method for marihuana samples used in amplified fragment length polymorphism ( AFLP ) analysis. J Forensic Sci. 48: 343-7.
- Miller-Coyle H. , S. Shulter, L. Tully, E. Pagliaro, A. Harper, T. Palmbach and, H.
C. Lee. 2005.
Validation of a DNA method for the individualisation of works grounds. hypertext transfer protocol: //www.ncjrs.gov/pdffiles1/nij/grants/211999.pdf
- Nakamura, G. R. 1969. Forensic facets of bladder stone hairs of Cannabis and other workss.
J Assoc Off Anal Chem. 52: 5-16
- Saunders, J. A. , M. J. Pedroni, L. Penrose and, T. Fist.
2001. AFLP DNA analysis of opium poppy. Crop Sci. 41: 1596-1601
- Siniscalco, G. , P.
Caputo and, S. Cozzolino. 1997. Ribosomal DNA analysis as a tool for the designation of Cannabis sativa L. specimens of forensic involvement. Science and Justice.
34: 171- 174.
- Stentz, M. 2009.
- Thornton, J. I. and G. R. Nakamura. 1972. The designation of marihuana.
J Forensic Sci Soc. 12: 461-519. United States Department of Justice.
2009. hypertext transfer protocol: //www.usdoj.gov/dea/statistics.html
- Vos, P. , R. Hogers, M.
Bleeker, M. Reijans, T. van de Lee and, M. Hornes. 1995.
AFLP: A new technique for DNA fingerprinting. Nucleic Acids Res. 23: 4407-4414
- Yoon, C. K. 1993.
Forensic Science: Botanical informant for the prosecution. Science. 260: 894-895.