- Email: [email protected]
Differentiation of Cannabis subspecies by THCA synthase gene analysis using RFLP
Accepted Manuscript Differentiation of Cannabis subspecies by THCA synthase gene analysis using RFLP analysis Natasa Cirovic, Miljana Kecmanovic, Dusan Keckarevic, Milica Keckarevic Markovic PII:
S1752-928X(17)30104-X
DOI:
10.1016/j.jflm.2017.07.015
Reference:
YJFLM 1524
To appear in:
Journal of Forensic and Legal Medicine
Received Date: 23 January 2017 Revised Date:
30 June 2017
Accepted Date: 24 July 2017
Please cite this article as: Cirovic N, Kecmanovic M, Keckarevic D, Keckarevic Markovic M, Differentiation of Cannabis subspecies by THCA synthase gene analysis using RFLP analysis, Journal of Forensic and Legal Medicine (2017), doi: 10.1016/j.jflm.2017.07.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Differentiation of Cannabis subspecies by THCA synthase gene analysis using RFLP analysis
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Natasa Cirovic1,2, Miljana Kecmanovic1,3, Dusan Keckarevic1,4, Milica Keckarevic Markovic1 University of Belgrade, Faculty of Biology, Studentski trg 3, Belgrade, Serbia
2
[email protected]
3
[email protected]
4
[email protected]
Corresponding author: Milica Keckarevic Markovic
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1
University of Belgrade, Faculty of Biology
11000 Belgrade Serbia
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Tel/fax: +381 11 2639100
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Studentski trg 3
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E-mail: [email protected]
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
ACCEPTED MANUSCRIPT Differentiation of Cannabis subspecies by RFLP analysis of THCA synthase gene
ABSTRACT Cannabis sativa subspecies, known as industrial hemp (C. sativa sativa) and marijuana
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(C. sativa indica) show no evident morphological distinctions, but they contain different levels of psychoactive ∆-9-tetrahidrocanabinol (THC), with considerably higher concentration in marijuana than in hemp. C. sativa subspecies differ in sequence of tetrahydrocannabinolic
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acid (THCA) synthase gene, responsible for THC production, and only one active copy of the gene, distinctive for marijuana, is capable of producing THC in concentration more then 0,3%
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in dried plants, usually punishable by the law.
Twenty different samples of marijuana that contain THC in concentration more then 0,3% and three varieties of industrial hemp were analyzed for presence of an active copy of THCA synthase gene using in-house developed restriction fragment length polymorphism
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(RFLP) method All twenty samples of marijuana were positive for the active copy of THCA synthase gene, 16 of them heterozygous. All three varieties of industrial hemp were homozygous for inactive copy.
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An algorithm for the fast and accurate forensic analysis of samples suspected to be marijuana was constructed, answering the question if an analyzed sample is capable of
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producing THC in concentrations higher than 0.3%.
KEYWORDS
Cannabis, hemp, false-negative, THC, RFLP, algorithm
1
ACCEPTED MANUSCRIPT INTRODUCTION Cannabis sativa ssp. sativa (hemp) is a traditional crop worldwide. Cannabis sativa ssp. indica (marijuana) is, on the other hand, subject of drug abuse. Both subspecies show no evident morphological distinctions, but they contain different levels of psychoactive
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substances, among them the most important ∆-9-tetrahidrocanabinol (THC), with considerably higher concentration in marijuana than in hemp1. The presence of psychoactive substances does not have any influence on the quality of plant fiber, but in certain
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circumstances one triggers the question whether the cultivated plant is harmless hemp, or potentially harmfull marijuana and whether the cultivation is acceptable. Possessing, growing
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and consuming marijuana is illegal in many world countries, while the production of industrial hemp is desirable. Cultivated variants of industrial hemp have higher disease and pest resistance, while at the same time they have psychoactive potential in acceptable boundaries2. Research of Cannabis sp. in forensic studies usually has a goal of making the
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disctincion between the subspecies of Cannabis sativa, i.e. the discrimination of C. sativa ssp. indica and C. sativa ssp. sativa.
Many jurisdictions have an upper limit of 0,2% or 0,3% for ∆-9-tetrahydrocannabinol
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(THC) content in dried plants. In Serbia, growing and possessing of plants capable of producing THC concentrations higher than 0.3% (as in marijuana) is considered punishable
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by the law.
Standard methodology for determining the content of THC in a sample is gas-
chromatography. The literature frequently cites few other methodologies based on High Performance Liquid Chromatography (HPLC). However, these methodologies have low sensitivity when separating different chemical compounds that are important in forensic analysis (such as THC, cannabinol (CBN), cannabidiol (CBD) etc.). Furthermore, when applying such methodologies, there is a risk of obtaining false negative results in the analysis 2
ACCEPTED MANUSCRIPT due to different plant age, sampling from different parts of the plant or as a result of decomposition of THC in the sample over time3. C. sativa subspecies differ in sequence of tetrahydrocannabinolic acid (THCA) synthase gene and its activity. THCA synthase gene is coding for an enzyme that catalyzes
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oxidative cyclization of cannabigerolic acid (CBGA) into tetrahydrocannabinol acid (THCA), which is then decarboxylatedinto THC by heating4. C. sativa ssp. sativa is homozigous for inactive variant of THCA synthase gene, which results in no production (or production on the
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very low level) of THC molecule in this plants. On the other hand, Cannabis sativa ssp. indica could be heterozygous for inactive/active or homozygous for active variant. The
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existence of at least one copy of the active gene enables production of THC in significant quantities5.
Recent reasearch is usually based on the analysis of THCA synthase gene6,7. There is a total of 63 nucleotide sequence variants (NSVs) that differentiate active and inactive copy of
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THCA synthase gene, which correspond to 37 amino acid substitutions in the THCA synthase8. Rotherham and Harbison developed snap-shot multiplex assay in which they analyzed four variants in 399 bp long sequence of THCA synthase gene5, while
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Sutipatanasomboon and Panvisavas developed duplex PCR test for C. sativa drug- and fibertype detection and discrimination9. We developed in house methods, based on allele specific
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PCR and on PCR and restriction digestion, to differentiate THCA synthase gene sequence in C. sativa subspecies.
The objective of this paper was to establish an algorithm for forensic DNA analysis in
order to determine if a plant in question is capable of producing THC in concentration higher than 0.3%, i.e. if it belongs to marijuana or „drug-type“ of cannabis, by using fast, simple and inexpensive method for the analysis of THCA synthase gene.
3
ACCEPTED MANUSCRIPT MATERIAL AND METHODS As a starting material the leaves and dried parts of C. sativa were used. The leaves from oak (Quercus robur) and tulip (Tulipa sp.) were used as controll samples. The fresh oak and tulip samples were obtained from a botanical garden, and the same day DNA isolation
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was conducted.
C. sativa ssp. sativa leaves were two days old. Three different hemp varieties from three different countries (USO11 from Ukraine, Fedora 17 from France and Novosadska from
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Serbia) were used as to have varied and genetically informative sample set.
Twenty dried samples of C. sativa ssp. indica (quantities from 13.1 to 125.4 mg) were
content greater then 0.3% by HPLC.
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brought for forensic DNA analysis. All samples were previously found positive for THC
DNA from C. sativa and control plants was isolated using ZR Plant/Seed DNA MiniPrep kit (ZymoResearch, USA) according to manufacturer`s protocol. Quality and
bromide (EtBr).
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quantity of isolated DNA was analysed by agarose gel electrophoresis, stained by ethidium
In order to identify C. sativa subspecies and, if applicable, plant origin of analyzed
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samples, we performed PCR and restriction digestion (Table 1). In general, three different PCR amplifications were conducted. By combining primers THCA/G-F THCA/F2-R
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fragment specific for marijuana THCA-synthase gene (of 277 bp) was amplified. PCR with the combination of THCA/E2-R THCA/C2-Fd primers resulted in a fragment of 400 bp, present both in marijuana and hemp. In 3rd PCR, we confirmed herbal origin of the material by amplification of 350 bp fragment of ribulose-1,5-bisphosphate carboxylase/oxygenase gene (RuBisCO, rbcL gene), part of chloroplast genome. 400 bp fragments of THCA synthase gene were subjected to overnight restriction digestion at 37⁰C, using enzyme RsaI (recognizes and cuts GTAC sequence on DNA). 4
ACCEPTED MANUSCRIPT RESULTS AND DISCUSSION All samples analyzed, including oak and tulip, were positive for the presence of rbcL gene, confirming their plant origin. 400 bp long PCR fragment was amplified in both cannabis subspecies under the
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stringent conditions (Table 1). When using less stringent annealing temperature (50⁰C), the inactive gene copy was preferentially amplified, probabily due to differences in nucleotide sequences that lie adjacent to primer binding sites. The other allele of the same gene
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(marijuana-specific fragment – active gene copy ) was amplified when using annealing temperature of 65⁰ C.
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After restriction digestion, different restriction maps for marijuana and industrial hemp were obtained. The result of such digestion were fragments of 211 bp and 189 bp in inactive variant and fragments of 211, 126 and 63 bp in active variant of THCA synathase gene (Tables 2 and 3). All samples of C. sativa ssp. sativa showed homozigosity for inactive
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variant, while Cannabis sativa ssp. indica were either heterozygous for inactive/active or homozygous for active variant of THCA synthase gene. After restriction digestion and agarose gel electrophoresis, 4 of 20 (20%) marijuana
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samples displayed three bands of 211, 126 i 63 bp, and 16 of 20 (80%) marijuana samples
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presented four bands of 211, 189, 126 and 63 bp. All three hemp varieties displayed two bands of 211 and 189 bp (Figure 1). Further, samples were subjected to allele specific PCR that produce fragments of
277 bp, aimed at amplification exclusively in marijuana samples (Table 4). Samples that had PCR/RD profile as C. sativa ssp. indica were amplified and produced 277 bp PCR product. Furthermore, in samples that had PCR/RD profile as C. sativa ssp. sativa, 277 bp PCR product was not amplified. Sequence variations were confirmed by Sanger sequencing of
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ACCEPTED MANUSCRIPT amplified fragments. Oak and tulip were negative for PCR amplification of both C. sativa and C. sativa ssp. indica specific fragments. In most cases, forensic analysis of suspected marijuana is based on presence of THC in confiscated material, but applied methodology could lead to false negative results, as
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consequence of a different plant age, sampling from different parts of the plant, or as a result of decomposition of THC in the sample over time5. On the other hand, in countries where growing and possession of marijuana is prohibited, the legal issue is if the analyzed sample
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belongs to a subspecies that is capable of producing THC in concentrations higher than 0,2% or 0,3%, depending on jurisdiction.
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So, especially in cases where THC content analyses have shown values less than the 0,2 or 0,3%, DNA analysis of THCA synathase gene, responsible for the last enzyme catalyzed step in THC production in Cannabis, should be done.
The methodology established is specific, based on analysis of the gene exclusively
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present in Cannabis species, and, on the other hand, based on differences in nucleotide sequence (NSV, nucleotide sequence variants) of that gene, which makes it suitable for Cannabis subspecies diversification.
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After the summary of results, an algorithm for the fast and accurate forensic analysis
2):
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of samples suspected to be marijuana is constructed, as presented in the figure below (Figure
400 bp long fragments of THCA synthase gene are amplified and then subjected to
restriction digestion, yielding three possible different digestion patterns. Two of them would confirm marijuana (fragments of length 211, 189, 126 and 63 bp or fragments of length 211, 126 and 63 bp) while the third one is specific for hemp (fragments of length 211 and 189 bp). When amplification is absent (or for a confirmation of PCR/RD analysis), a shorter THCA synthase gene fragment, 277 bp long, should be amplified. A positive reaction would confirm 6
ACCEPTED MANUSCRIPT that the analyzed sample is marijuana, while a negative reaction would implicate necessity for further analysis. If so, the sample should be tested for presence of a 350 bp long sequence of rbcL gene. If rbcL sequence is detected, the conclusion should be that the sample is of herbal origin, but not marijuana. Otherwise, the sample should be considered non-herbal,
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significantly degraded or inhibited, in large, nonsuitable for analyses. CONCLUSION
An algorithm for the fast and accurate forensic analysis of samples suspected to be
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marijuana was constructed, answering the question if an analyzed sample is capable of producing significant quantities of THC (concentrations higher than 0.3% in dried plants, as
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specified by Serbian legislative) and to overcome the problem that could arise from false – negative results due to different plant age, sampling from different parts of the plant or as a
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result of decomposition of THC in the sample.
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ACCEPTED MANUSCRIPT REFERENCES: 1. Kojoma M, Iida O, Makino Y, Satake M, Sekita S (2002) DNA Fingerprinting of Cannabis sativa Using Inter-Simple Sequence Repeat (ISSR) Amplification. Planta Med 68: 60-63.
plants of hemp varieties. Ind Crops Prod 19: 19-24.
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2. Mechtler K, Bailer J, De Huber K (2004) Variations of ∆9-THC content in single
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3. United Nations Publication (2009) Recommended Methods for the Identification and Analysis of Cannabis and Cannabis Products. Manual for use by national drug analysis
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laboratories. Sales No.E.09.XI.15. ISBN 978-92-1-148242-3.
4. Taura F, Sirikantaramas S, Shoyama Y, Yoshikai K, Morimoto S, Shoyama Y (2007) Cannabidioloc-acid synthase, the chemotype-determining enzyme in the fiber-type Cannabis sativa. FEBS Lett 581(16): 2929-2934.
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5. Rotherham D, Harbison SA (2011) Differentiation of drug and non-drug Cannabis using a single nucleotide polymorphism (SNP) assay. Forensic Sci Int 207:193-197. 6. Datwyler SL, Weiblen GD (2006) Genetic variation in hemp and marijuana (Cannabis
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sativa L.) according to amplified fragment lenght polymorphisms. J Forensic Sci
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51(2): 371-375.
7. Sirikantaramas S, Morimoto S, Shoyama Y, Ishikawa Y, Wada Y, Shoyama Y, et al (2004) The gene controlling marijuana psychoactivity. J Biol. Chem 279: 3976739774.
8. Kojoma M, Seki H, Yoshida S, Muranaka T (2006) DNA polymorphisms in the tetrahydrocannabinolic acid (THCA) synthase gene in ’drug-type’ and ’fiber-type’ Cannabis sativa L. Forensic Sci Int 159: 132-140. 8
ACCEPTED MANUSCRIPT 9. Sutipatanasomboon A, Panvisavas N (2011) Discrimination of ‘fiber-type’ and ‘drugtype’ Cannabis sativa L.by fluorescent duplex PCR. Forensic Sci Int: Genetics Supplement Series 3: e522–e523. 10. Hasebe M, Omori T, Nakazawa M,. Sano T, Kato M, Iwatsuki K (1994) RbcL gene
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Proc Nat Acad Sci USA 91: 5730-5734.
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sequences provide evidence for the evolutionary lineages of leptsporangiate ferns.
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Fig 1: Restriction profiles of C. sativa ssp. THCA synthase genes: Lane 1: 100 bp DNA ladder (GeneRulerTM, Fermentas, Waltham, MA, SAD), lane 2: marijuana sample heterozygous for THCA synthase gene (visible bands of 63, 126, 189 and 211 bp), lanes 3 and 4: marijuana samples homozygous for active THCA synthase gene (63, 126, and 211 bp), lane 5: industrial hemp sample homozygous for inactive THCA synthase gene (189 and 211 bp).
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Fig 2: The algorithm for forensic analysis of a material suspected to be marijuana
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Primer sequence
Specificity
THCA/G-F THCA/F2-R
5’-AATAACTCCCATATCCAAGCA-3’ (8) 5’-TATTGCCCTACTGTTGGCG-3’ (8)
Marijuana
THCA/E2-R
5’-CGTCTTCTTCCCAGCTGATC-3’ (8) 5’-CAAACTGGTTGCTGTCCCATC-3’ (8)
RbcL/sF RbcL/cR
5’-ACTGTAGTGGGCAAATTGGAAGGCGAACG-3’ (11) 5’-GCAGCAGCTAGTTCCGGGCTCCA-3’ (11)
Plants
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THCA/C2-Fd
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Table 1. The overview of PCR and restriction digestion analysis, NA – not applicable
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Product lengths
Product lengths after RsaI restriction digestion
50⁰C
277 bp
NA
65⁰C
400 bp
Active THCA synthase gene 63, 126 and 211 bp Inactive THCA synthase gene 189 and 211 bp
50⁰C
350 bp
NA
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Marijuana and hemp
Annealing temperature
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Primer name
ACCEPTED MANUSCRIPT CAAACTGGTTGCTGTCCCATCAAAGTCTACTATATTCAGTGTTAAAAAGAACATGGAGATACATGGGC TTGTCAAGTTATTTAACAAATGGCAAAATATTGCTTACAAGTATGACAAAGATTTAGTACTCATGACT CACTTCATAACAAAGAATATTACAGATAATCATGGGAAGAATAAGACTACAGTACATGGTTACTTCTC TTCAATTTTTCATGGTGGAGTGGATAGTCTAGTCGACTTGATGAACAAGAGCTTTCCTGAGTTGGGTA TTAAAAAAACTGATTGCAAAGAATTTAGCTGGATTGATACAACCATCTTCTACAGTGGTGTTGTAAAT TTTAACACTGCTAATTTTAAAAAGGAAATTTTGCTTGATAGATCAGCTGGGAAGAAGACG
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Table 2. 400 bp nucleotide sequence of active THCA synthase gene amplified with primers THCA/C2-Fd and THCA/E2-R (Table 1) (GenBank: AB212832.1, Kojoma et al., 2006, c.738-1137 bp). The restriction sites are highlighted
13
ACCEPTED MANUSCRIPT
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CAAACTTGTTGTTGTCCCATCAAAGGCTACTATATTCAGTGTTAAAAAGAACATGGAGATACATGGGC TTGTCAAGTTATTTAACAAATGGCAAAATATTGCTTACAAGTATGACAAAGATTTAATGCTCACGACT CACTTCAGAACTAGGAATATTACAGATAATCATGGGAAGAATAAGACTACAGTACATGGTTACTTCTC TTCCATTTTTCTTGGTGGAGTGGATAGTCTAGTTGACTTGATGAACAAGAGCTTTCCTGAGTTGGGTA TTAAAAAAACTGATTGCAAAGAATTGAGCTGGATTGATACAACCATCTTCTACAGTGGTGTTGTAAAT TACAACACTGCTAATTTTAAAAAGGAAATTTTGCTTGATAGATCAGCTGGGAAGAAGACG
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Table 3. 400 bp nucleotide sequence of inactive THCA synthase gene amplified with primers THCA/C2-Fd and THCA/E2-R (Table 1) (GenBank: AB212840.1, Kojoma et al., 2006, c.7381137 bp). The restriction sites are highlighted.
14
ACCEPTED MANUSCRIPT C. sativa ssp. sativa AATGTCTCCCATATCCAGGCCAGTAT TCTCTGCTCCAAGAAAGTTGGTTTGC AGATTCGAACTCGAAGCGGTGGCCAT GATGCTGAGGGTTTGTCCTACATATC TCAAGTCCCATTTGCTATAGTAGACT TGAGAAACATGCATACGGTCAAAGT AGATATTCATAGCCAAACTGCGTGGG TTGAAGCCGGAGCTACCCTTGGAGAA GTTTATTATTGGATCAATGAGATGAA TGAGAATTTTAGTTTTCCTGGTGGGT ATTGCCCTACTGTTGGCG
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C. sativa ssp. indica AATAACTCCCATATCCAAGCAACTATT TTATGCTCTAAGAAAGTTGGCTTGCAGA TTCGAACTCGAAGCGGTGGCCATGATG CTGAGGGTATGTCCTACATATCTCAAGT CCCATTTGTTGTAGTAGACTTGAGAAAC ATGCATTCGATCAAAATAGATGTTCATA GCCAAACTGCGTGGGTTGAAGCCGGAG CTACCCTTGGAGAAGTTTATTATTGGAT CAATGAGAAGAATGAGAATCTTAGTTT TCCTGGTGGGTATTGCCCTACTGTTGG CG
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Table 4. 277 nucleotide sequence of THCA-synthase gen in marijuana and hemp. (primers THCA/G-F and THCA/F2-R (Table 1) (GenBank: AB212832.1 and AB212840.1, respectively, Kojoma et al., 2006, c.265-541 bp) Primers (THCA/G-F matches only the marijuana sequence) are highlighted. The differences between THCA/G-F primer and corresponding sequence in hemp (underlined) is red.
15
ACCEPTED MANUSCRIPT
AKNOWLEDGEMENTS C. sativa ssp. sativa specimens were kindly provided by Prof. d. Janos Bereni from the
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University of Novi Sad, Serbia.
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HIGHLIGHTS An algorythm for forensic analysis of marijuana was created
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The method adresses possible false-negative results
•
The method is based on nucleotide sequence variants in THCA synthase gene
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•
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This research did not receive any specific grant from funding agencies in the public, commercial,
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or not-for-profit sectors.
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CONFLICT OF INTEREST
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The authors declare that they have no conflict of interest.
S1752-928X(17)30104-X
DOI:
10.1016/j.jflm.2017.07.015
Reference:
YJFLM 1524
To appear in:
Journal of Forensic and Legal Medicine
Received Date: 23 January 2017 Revised Date:
30 June 2017
Accepted Date: 24 July 2017
Please cite this article as: Cirovic N, Kecmanovic M, Keckarevic D, Keckarevic Markovic M, Differentiation of Cannabis subspecies by THCA synthase gene analysis using RFLP analysis, Journal of Forensic and Legal Medicine (2017), doi: 10.1016/j.jflm.2017.07.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Differentiation of Cannabis subspecies by THCA synthase gene analysis using RFLP analysis
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Natasa Cirovic1,2, Miljana Kecmanovic1,3, Dusan Keckarevic1,4, Milica Keckarevic Markovic1 University of Belgrade, Faculty of Biology, Studentski trg 3, Belgrade, Serbia
2
[email protected]
3
[email protected]
4
[email protected]
Corresponding author: Milica Keckarevic Markovic
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1
University of Belgrade, Faculty of Biology
11000 Belgrade Serbia
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Tel/fax: +381 11 2639100
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Studentski trg 3
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E-mail: [email protected]
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
ACCEPTED MANUSCRIPT Differentiation of Cannabis subspecies by RFLP analysis of THCA synthase gene
ABSTRACT Cannabis sativa subspecies, known as industrial hemp (C. sativa sativa) and marijuana
RI PT
(C. sativa indica) show no evident morphological distinctions, but they contain different levels of psychoactive ∆-9-tetrahidrocanabinol (THC), with considerably higher concentration in marijuana than in hemp. C. sativa subspecies differ in sequence of tetrahydrocannabinolic
SC
acid (THCA) synthase gene, responsible for THC production, and only one active copy of the gene, distinctive for marijuana, is capable of producing THC in concentration more then 0,3%
M AN U
in dried plants, usually punishable by the law.
Twenty different samples of marijuana that contain THC in concentration more then 0,3% and three varieties of industrial hemp were analyzed for presence of an active copy of THCA synthase gene using in-house developed restriction fragment length polymorphism
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(RFLP) method All twenty samples of marijuana were positive for the active copy of THCA synthase gene, 16 of them heterozygous. All three varieties of industrial hemp were homozygous for inactive copy.
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An algorithm for the fast and accurate forensic analysis of samples suspected to be marijuana was constructed, answering the question if an analyzed sample is capable of
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producing THC in concentrations higher than 0.3%.
KEYWORDS
Cannabis, hemp, false-negative, THC, RFLP, algorithm
1
ACCEPTED MANUSCRIPT INTRODUCTION Cannabis sativa ssp. sativa (hemp) is a traditional crop worldwide. Cannabis sativa ssp. indica (marijuana) is, on the other hand, subject of drug abuse. Both subspecies show no evident morphological distinctions, but they contain different levels of psychoactive
RI PT
substances, among them the most important ∆-9-tetrahidrocanabinol (THC), with considerably higher concentration in marijuana than in hemp1. The presence of psychoactive substances does not have any influence on the quality of plant fiber, but in certain
SC
circumstances one triggers the question whether the cultivated plant is harmless hemp, or potentially harmfull marijuana and whether the cultivation is acceptable. Possessing, growing
M AN U
and consuming marijuana is illegal in many world countries, while the production of industrial hemp is desirable. Cultivated variants of industrial hemp have higher disease and pest resistance, while at the same time they have psychoactive potential in acceptable boundaries2. Research of Cannabis sp. in forensic studies usually has a goal of making the
TE D
disctincion between the subspecies of Cannabis sativa, i.e. the discrimination of C. sativa ssp. indica and C. sativa ssp. sativa.
Many jurisdictions have an upper limit of 0,2% or 0,3% for ∆-9-tetrahydrocannabinol
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(THC) content in dried plants. In Serbia, growing and possessing of plants capable of producing THC concentrations higher than 0.3% (as in marijuana) is considered punishable
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by the law.
Standard methodology for determining the content of THC in a sample is gas-
chromatography. The literature frequently cites few other methodologies based on High Performance Liquid Chromatography (HPLC). However, these methodologies have low sensitivity when separating different chemical compounds that are important in forensic analysis (such as THC, cannabinol (CBN), cannabidiol (CBD) etc.). Furthermore, when applying such methodologies, there is a risk of obtaining false negative results in the analysis 2
ACCEPTED MANUSCRIPT due to different plant age, sampling from different parts of the plant or as a result of decomposition of THC in the sample over time3. C. sativa subspecies differ in sequence of tetrahydrocannabinolic acid (THCA) synthase gene and its activity. THCA synthase gene is coding for an enzyme that catalyzes
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oxidative cyclization of cannabigerolic acid (CBGA) into tetrahydrocannabinol acid (THCA), which is then decarboxylatedinto THC by heating4. C. sativa ssp. sativa is homozigous for inactive variant of THCA synthase gene, which results in no production (or production on the
SC
very low level) of THC molecule in this plants. On the other hand, Cannabis sativa ssp. indica could be heterozygous for inactive/active or homozygous for active variant. The
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existence of at least one copy of the active gene enables production of THC in significant quantities5.
Recent reasearch is usually based on the analysis of THCA synthase gene6,7. There is a total of 63 nucleotide sequence variants (NSVs) that differentiate active and inactive copy of
TE D
THCA synthase gene, which correspond to 37 amino acid substitutions in the THCA synthase8. Rotherham and Harbison developed snap-shot multiplex assay in which they analyzed four variants in 399 bp long sequence of THCA synthase gene5, while
EP
Sutipatanasomboon and Panvisavas developed duplex PCR test for C. sativa drug- and fibertype detection and discrimination9. We developed in house methods, based on allele specific
AC C
PCR and on PCR and restriction digestion, to differentiate THCA synthase gene sequence in C. sativa subspecies.
The objective of this paper was to establish an algorithm for forensic DNA analysis in
order to determine if a plant in question is capable of producing THC in concentration higher than 0.3%, i.e. if it belongs to marijuana or „drug-type“ of cannabis, by using fast, simple and inexpensive method for the analysis of THCA synthase gene.
3
ACCEPTED MANUSCRIPT MATERIAL AND METHODS As a starting material the leaves and dried parts of C. sativa were used. The leaves from oak (Quercus robur) and tulip (Tulipa sp.) were used as controll samples. The fresh oak and tulip samples were obtained from a botanical garden, and the same day DNA isolation
RI PT
was conducted.
C. sativa ssp. sativa leaves were two days old. Three different hemp varieties from three different countries (USO11 from Ukraine, Fedora 17 from France and Novosadska from
SC
Serbia) were used as to have varied and genetically informative sample set.
Twenty dried samples of C. sativa ssp. indica (quantities from 13.1 to 125.4 mg) were
content greater then 0.3% by HPLC.
M AN U
brought for forensic DNA analysis. All samples were previously found positive for THC
DNA from C. sativa and control plants was isolated using ZR Plant/Seed DNA MiniPrep kit (ZymoResearch, USA) according to manufacturer`s protocol. Quality and
bromide (EtBr).
TE D
quantity of isolated DNA was analysed by agarose gel electrophoresis, stained by ethidium
In order to identify C. sativa subspecies and, if applicable, plant origin of analyzed
EP
samples, we performed PCR and restriction digestion (Table 1). In general, three different PCR amplifications were conducted. By combining primers THCA/G-F THCA/F2-R
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fragment specific for marijuana THCA-synthase gene (of 277 bp) was amplified. PCR with the combination of THCA/E2-R THCA/C2-Fd primers resulted in a fragment of 400 bp, present both in marijuana and hemp. In 3rd PCR, we confirmed herbal origin of the material by amplification of 350 bp fragment of ribulose-1,5-bisphosphate carboxylase/oxygenase gene (RuBisCO, rbcL gene), part of chloroplast genome. 400 bp fragments of THCA synthase gene were subjected to overnight restriction digestion at 37⁰C, using enzyme RsaI (recognizes and cuts GTAC sequence on DNA). 4
ACCEPTED MANUSCRIPT RESULTS AND DISCUSSION All samples analyzed, including oak and tulip, were positive for the presence of rbcL gene, confirming their plant origin. 400 bp long PCR fragment was amplified in both cannabis subspecies under the
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stringent conditions (Table 1). When using less stringent annealing temperature (50⁰C), the inactive gene copy was preferentially amplified, probabily due to differences in nucleotide sequences that lie adjacent to primer binding sites. The other allele of the same gene
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(marijuana-specific fragment – active gene copy ) was amplified when using annealing temperature of 65⁰ C.
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After restriction digestion, different restriction maps for marijuana and industrial hemp were obtained. The result of such digestion were fragments of 211 bp and 189 bp in inactive variant and fragments of 211, 126 and 63 bp in active variant of THCA synathase gene (Tables 2 and 3). All samples of C. sativa ssp. sativa showed homozigosity for inactive
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variant, while Cannabis sativa ssp. indica were either heterozygous for inactive/active or homozygous for active variant of THCA synthase gene. After restriction digestion and agarose gel electrophoresis, 4 of 20 (20%) marijuana
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samples displayed three bands of 211, 126 i 63 bp, and 16 of 20 (80%) marijuana samples
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presented four bands of 211, 189, 126 and 63 bp. All three hemp varieties displayed two bands of 211 and 189 bp (Figure 1). Further, samples were subjected to allele specific PCR that produce fragments of
277 bp, aimed at amplification exclusively in marijuana samples (Table 4). Samples that had PCR/RD profile as C. sativa ssp. indica were amplified and produced 277 bp PCR product. Furthermore, in samples that had PCR/RD profile as C. sativa ssp. sativa, 277 bp PCR product was not amplified. Sequence variations were confirmed by Sanger sequencing of
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consequence of a different plant age, sampling from different parts of the plant, or as a result of decomposition of THC in the sample over time5. On the other hand, in countries where growing and possession of marijuana is prohibited, the legal issue is if the analyzed sample
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belongs to a subspecies that is capable of producing THC in concentrations higher than 0,2% or 0,3%, depending on jurisdiction.
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So, especially in cases where THC content analyses have shown values less than the 0,2 or 0,3%, DNA analysis of THCA synathase gene, responsible for the last enzyme catalyzed step in THC production in Cannabis, should be done.
The methodology established is specific, based on analysis of the gene exclusively
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present in Cannabis species, and, on the other hand, based on differences in nucleotide sequence (NSV, nucleotide sequence variants) of that gene, which makes it suitable for Cannabis subspecies diversification.
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After the summary of results, an algorithm for the fast and accurate forensic analysis
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of samples suspected to be marijuana is constructed, as presented in the figure below (Figure
400 bp long fragments of THCA synthase gene are amplified and then subjected to
restriction digestion, yielding three possible different digestion patterns. Two of them would confirm marijuana (fragments of length 211, 189, 126 and 63 bp or fragments of length 211, 126 and 63 bp) while the third one is specific for hemp (fragments of length 211 and 189 bp). When amplification is absent (or for a confirmation of PCR/RD analysis), a shorter THCA synthase gene fragment, 277 bp long, should be amplified. A positive reaction would confirm 6
ACCEPTED MANUSCRIPT that the analyzed sample is marijuana, while a negative reaction would implicate necessity for further analysis. If so, the sample should be tested for presence of a 350 bp long sequence of rbcL gene. If rbcL sequence is detected, the conclusion should be that the sample is of herbal origin, but not marijuana. Otherwise, the sample should be considered non-herbal,
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significantly degraded or inhibited, in large, nonsuitable for analyses. CONCLUSION
An algorithm for the fast and accurate forensic analysis of samples suspected to be
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marijuana was constructed, answering the question if an analyzed sample is capable of producing significant quantities of THC (concentrations higher than 0.3% in dried plants, as
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specified by Serbian legislative) and to overcome the problem that could arise from false – negative results due to different plant age, sampling from different parts of the plant or as a
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result of decomposition of THC in the sample.
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ACCEPTED MANUSCRIPT REFERENCES: 1. Kojoma M, Iida O, Makino Y, Satake M, Sekita S (2002) DNA Fingerprinting of Cannabis sativa Using Inter-Simple Sequence Repeat (ISSR) Amplification. Planta Med 68: 60-63.
plants of hemp varieties. Ind Crops Prod 19: 19-24.
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2. Mechtler K, Bailer J, De Huber K (2004) Variations of ∆9-THC content in single
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3. United Nations Publication (2009) Recommended Methods for the Identification and Analysis of Cannabis and Cannabis Products. Manual for use by national drug analysis
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laboratories. Sales No.E.09.XI.15. ISBN 978-92-1-148242-3.
4. Taura F, Sirikantaramas S, Shoyama Y, Yoshikai K, Morimoto S, Shoyama Y (2007) Cannabidioloc-acid synthase, the chemotype-determining enzyme in the fiber-type Cannabis sativa. FEBS Lett 581(16): 2929-2934.
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5. Rotherham D, Harbison SA (2011) Differentiation of drug and non-drug Cannabis using a single nucleotide polymorphism (SNP) assay. Forensic Sci Int 207:193-197. 6. Datwyler SL, Weiblen GD (2006) Genetic variation in hemp and marijuana (Cannabis
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sativa L.) according to amplified fragment lenght polymorphisms. J Forensic Sci
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51(2): 371-375.
7. Sirikantaramas S, Morimoto S, Shoyama Y, Ishikawa Y, Wada Y, Shoyama Y, et al (2004) The gene controlling marijuana psychoactivity. J Biol. Chem 279: 3976739774.
8. Kojoma M, Seki H, Yoshida S, Muranaka T (2006) DNA polymorphisms in the tetrahydrocannabinolic acid (THCA) synthase gene in ’drug-type’ and ’fiber-type’ Cannabis sativa L. Forensic Sci Int 159: 132-140. 8
ACCEPTED MANUSCRIPT 9. Sutipatanasomboon A, Panvisavas N (2011) Discrimination of ‘fiber-type’ and ‘drugtype’ Cannabis sativa L.by fluorescent duplex PCR. Forensic Sci Int: Genetics Supplement Series 3: e522–e523. 10. Hasebe M, Omori T, Nakazawa M,. Sano T, Kato M, Iwatsuki K (1994) RbcL gene
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Proc Nat Acad Sci USA 91: 5730-5734.
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sequences provide evidence for the evolutionary lineages of leptsporangiate ferns.
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Fig 1: Restriction profiles of C. sativa ssp. THCA synthase genes: Lane 1: 100 bp DNA ladder (GeneRulerTM, Fermentas, Waltham, MA, SAD), lane 2: marijuana sample heterozygous for THCA synthase gene (visible bands of 63, 126, 189 and 211 bp), lanes 3 and 4: marijuana samples homozygous for active THCA synthase gene (63, 126, and 211 bp), lane 5: industrial hemp sample homozygous for inactive THCA synthase gene (189 and 211 bp).
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Fig 2: The algorithm for forensic analysis of a material suspected to be marijuana
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Primer sequence
Specificity
THCA/G-F THCA/F2-R
5’-AATAACTCCCATATCCAAGCA-3’ (8) 5’-TATTGCCCTACTGTTGGCG-3’ (8)
Marijuana
THCA/E2-R
5’-CGTCTTCTTCCCAGCTGATC-3’ (8) 5’-CAAACTGGTTGCTGTCCCATC-3’ (8)
RbcL/sF RbcL/cR
5’-ACTGTAGTGGGCAAATTGGAAGGCGAACG-3’ (11) 5’-GCAGCAGCTAGTTCCGGGCTCCA-3’ (11)
Plants
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THCA/C2-Fd
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Table 1. The overview of PCR and restriction digestion analysis, NA – not applicable
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Product lengths
Product lengths after RsaI restriction digestion
50⁰C
277 bp
NA
65⁰C
400 bp
Active THCA synthase gene 63, 126 and 211 bp Inactive THCA synthase gene 189 and 211 bp
50⁰C
350 bp
NA
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Marijuana and hemp
Annealing temperature
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Primer name
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Table 2. 400 bp nucleotide sequence of active THCA synthase gene amplified with primers THCA/C2-Fd and THCA/E2-R (Table 1) (GenBank: AB212832.1, Kojoma et al., 2006, c.738-1137 bp). The restriction sites are highlighted
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CAAACTTGTTGTTGTCCCATCAAAGGCTACTATATTCAGTGTTAAAAAGAACATGGAGATACATGGGC TTGTCAAGTTATTTAACAAATGGCAAAATATTGCTTACAAGTATGACAAAGATTTAATGCTCACGACT CACTTCAGAACTAGGAATATTACAGATAATCATGGGAAGAATAAGACTACAGTACATGGTTACTTCTC TTCCATTTTTCTTGGTGGAGTGGATAGTCTAGTTGACTTGATGAACAAGAGCTTTCCTGAGTTGGGTA TTAAAAAAACTGATTGCAAAGAATTGAGCTGGATTGATACAACCATCTTCTACAGTGGTGTTGTAAAT TACAACACTGCTAATTTTAAAAAGGAAATTTTGCTTGATAGATCAGCTGGGAAGAAGACG
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Table 3. 400 bp nucleotide sequence of inactive THCA synthase gene amplified with primers THCA/C2-Fd and THCA/E2-R (Table 1) (GenBank: AB212840.1, Kojoma et al., 2006, c.7381137 bp). The restriction sites are highlighted.
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ACCEPTED MANUSCRIPT C. sativa ssp. sativa AATGTCTCCCATATCCAGGCCAGTAT TCTCTGCTCCAAGAAAGTTGGTTTGC AGATTCGAACTCGAAGCGGTGGCCAT GATGCTGAGGGTTTGTCCTACATATC TCAAGTCCCATTTGCTATAGTAGACT TGAGAAACATGCATACGGTCAAAGT AGATATTCATAGCCAAACTGCGTGGG TTGAAGCCGGAGCTACCCTTGGAGAA GTTTATTATTGGATCAATGAGATGAA TGAGAATTTTAGTTTTCCTGGTGGGT ATTGCCCTACTGTTGGCG
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C. sativa ssp. indica AATAACTCCCATATCCAAGCAACTATT TTATGCTCTAAGAAAGTTGGCTTGCAGA TTCGAACTCGAAGCGGTGGCCATGATG CTGAGGGTATGTCCTACATATCTCAAGT CCCATTTGTTGTAGTAGACTTGAGAAAC ATGCATTCGATCAAAATAGATGTTCATA GCCAAACTGCGTGGGTTGAAGCCGGAG CTACCCTTGGAGAAGTTTATTATTGGAT CAATGAGAAGAATGAGAATCTTAGTTT TCCTGGTGGGTATTGCCCTACTGTTGG CG
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Table 4. 277 nucleotide sequence of THCA-synthase gen in marijuana and hemp. (primers THCA/G-F and THCA/F2-R (Table 1) (GenBank: AB212832.1 and AB212840.1, respectively, Kojoma et al., 2006, c.265-541 bp) Primers (THCA/G-F matches only the marijuana sequence) are highlighted. The differences between THCA/G-F primer and corresponding sequence in hemp (underlined) is red.
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AKNOWLEDGEMENTS C. sativa ssp. sativa specimens were kindly provided by Prof. d. Janos Bereni from the
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University of Novi Sad, Serbia.
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HIGHLIGHTS An algorythm for forensic analysis of marijuana was created
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The method adresses possible false-negative results
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The method is based on nucleotide sequence variants in THCA synthase gene
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This research did not receive any specific grant from funding agencies in the public, commercial,
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or not-for-profit sectors.
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CONFLICT OF INTEREST
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The authors declare that they have no conflict of interest.