Differentiation of Cannabis subspecies by THCA synthase gene analysis using RFLP

Highlights

• An algorithm for forensic analysis of marijuana was created.

• The method addresses possible false-negative results.

• The method is based on nucleotide sequence variants in THCA synthase gene.

Abstract

Cannabis sativa subspecies, known as industrial hemp (C. sativa sativa) and marijuana (C. sativa indica) show no evident morphological distinctions, but they contain different levels of psychoactive Δ-9-tetrahidrocanabinol (THC), with considerably higher concentration in marijuana than in hemp. C. sativa subspecies differ in sequence of tetrahydrocannabinolic 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% 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 (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.

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 producing THC in concentrations higher than 0.3%.

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 substances, among them the most important Δ-9-tetrahidrocanabinol (THC), with considerably higher concentration in marijuana than in hemp.¹ The presence of psychoactive substances does not have any influence on the quality of plant fiber, but in certain circumstances one triggers the question whether the cultivated plant is harmless hemp, or potentially harmfull marijuana and whether the cultivation is acceptable. Possessing, growing 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 boundaries.² Research of Cannabis sp. in forensic studies usually has a goal of making the 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 Δ-9-tetrahydrocannabinol (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 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 due to different plant age, sampling from different parts of the plant or as a result of decomposition of THC in the sample over time.³

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 oxidative cyclization of cannabigerolic acid (CBGA) into tetrahydrocannabinol acid (THCA), which is then decarboxylatedinto THC by heating.⁴ C. sativa ssp. sativa is homozigous for inactive variant of THCA synthase gene, which results in no production (or production on the 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 existence of at least one copy of the active gene enables production of THC in significant quantities.⁵

Recent reasearch is usually based on the analysis of THCA synthase gene.6, 7 There is a total of 63 nucleotide sequence variants (NSVs) that differentiate active and inactive copy of THCA synthase gene, which correspond to 37 amino acid substitutions in the THCA synthase.⁸ Rotherham and Harbison developed snap-shot multiplex assay in which they analyzed four variants in 399 bp long sequence of THCA synthase gene,⁵ while Sutipatanasomboon and Panvisavas developed duplex PCR test for C. sativa drug- and fiber-type detection and discrimination.⁹ We developed in house methods, based on allele specific 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.