Tom Gilbert, Editor

The extraction and purification of nucleic acids is the first step in most molecular biology analysis techniques. The objective of this work is to obtain highly purified nucleic acids derived from Cannabis sativa resin seizure in order to conduct a DNA typing method for the individualization of cannabis resin samples. To obtain highly purified nucleic acids from cannabis resin (Hashish) free from contaminants that cause inhibition of PCR reaction, we have tested two protocols: the CTAB protocol of Wagner and a CTAB protocol described by Somma (2004) adapted for difficult matrix. We obtained high quality genomic DNA from 8 cannabis resin seizures using the adapted protocol. DNA extracted by the Wagner CTAB protocol failed to give polymerase chain reaction (PCR) amplification of tetrahydrocannabinolic acid (THCA) synthase coding gene. However, the extracted DNA by the second protocol permits amplification of THCA synthase coding gene using different sets of primers as assessed by PCR. We describe here for the first time the possibility of DNA extraction from (Hashish) resin derived from Cannabis sativa. This allows the use of DNA molecular tests under special forensic circumstances.

Samples

Eight samples of cannabis resin (Hashish) seizure were provided by the Laboratory of Forensic Sciences of Moroccan Gendarmerie Royal [LARATES]. These samples were seized from the North of Morocco.

DNA extractions

We have tested two protocols for DNA extraction from the eight cannabis Hashishsamples. A Wagner CTAB genomic DNA isolation method [17] originated from the research of Murray and Thompson and Somma CTAB protocol adapted for GMO in food matrix [21]. The only parameter we changed in the adapted protocol described by Somma is the weight of the matrix used, 200 mg instead of 100 mg. All the others steps are kept as described in the protocol.

Yield and quality of nucleic acid extracts

DNA extracted by both protocols was quantified using NanoDrop 8000 spectrophotometer (Thermo). One absorbance unit (260 nm) was assumed to correspond to 50 ng of nucleic acid per µl of solution. The purity of the samples was estimated from the (A₂₆₀/A₂₈₀) ratio.

Repurification step

Since DNA extracted by the Wagner CTAB protocol was of poor quality, a re-purification step was necessary. The ethanol precipitation is used to concentrate and precipitating DNA in the presence of ammonium acetate as binding salt [22].

PCR amplification and detection

To check the suitability of extracted DNA using both protocols, a downstream analysis by PCR reaction was performed. To this end, we have chosen to amplify for the full-length coding region of the THC synthase gene using the following primers a/b as described previously [23](Table 1), and the amplification of different fragments of the gene using a combination of primers g/f; a/f; c/e; c/h; d/h; d/b (Table 1). The same combination of primers was used for sequencing in order to assemble the complete sequence of the THCA synthase gene. The Amplification was performed in a total volume of 25 µl, with each reaction containing, 1× reaction buffer (Invitrogen), 2.5 mMgCl2, 0.5× Q-Solution (Qiagen), 0.2 mM dNTPs, 0.4 µM of each primers, 0.04 U/µl of Platinum DNA polymerase (Invitrogen), and 20 ng/µl of template DNA. The PCR conditions were: preheating at 94°C for 5 min, 35 cycles at 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min with a final extension at 72°C for 10 min. The reactions were carried out in a BIO RAD S1000 Thermal Cycler.

Agarose gel electrophoresis

The amplified fragments were electrophoresed on 1% agarose gel and detected using ethidium bromide along with a molecular weight marker 1 kb (Invitrogen). The DNAs integrity of all samples extracted with both protocols was assessed using 0.8% agarose gel electrophoresis. The gels were visualised under UV trans-illuminator using the G: BOX gel documentation system (Syngene).

Sequence analysis

The amplified PCR products were purified using ExoSAP-IT PCR Purification Kit (Affymetrix) according to the manufacturer’s instructions. For each purified PCR product, we have performed a sequencing reaction with the forward and reverse primer a/b; g/f; a/f; c/e; c/h; d/h ;d/b. Sequencing reaction was carried out using the BigDye Terminator cycle Sequencing Kit v3.1 (Applied Biosystems) according to the manufacturers’ instructions. Automated DNA sequencing was performed using an Applied Biosystems 3130XL sequencer. Sequence data were assembled using DNA Baser Sequence Assembler[24].

Nucleotide sequence accession numbers

The THC synthase gene sequences determined in the present study has been deposited under the GenBank accession numbers: JQ437481-JQ437488.

Yield and quality of nucleic acid extracts

DNA extraction using Wagner CTAB protocol provided a yield ranging from 15.3 to 373.8 (ng/µl).This yield was about 40.88 to 303.4 (ng/µl) after an additional step of purification (Table 2).

With the Adapted CTAB protocol the yield was 24 to 424.2(ng/µl) (Table 2).

Concerning the quality of nucleic acids, Wagner CTAB protocol with an additional step of purification gives a value of the ratio (A₂₆₀/A₂₈₀) ranging from 1.49 to 1.88. This may be due to high levels of phenolic and polysaccharides in the samples as previously reported[25]. However this ratio (A₂₆₀/A₂₈₀) ranged from 1.9 to 2.15 with the second CTAB protocol, indicating high quality nucleic acids extracted. On the other hand, the ratio (A₂₆₀/A₂₃₀) was also used as a secondary measurement of nucleic acids purity. The standard value of this ratio (A₂₆₀/A₂₃₀) ranges from 2.0 to 2.2.The Adapted CTAB protocol gives the best values of the (A₂₆₀/A₂₃₀) ratio. However, this ratio was lower than 2 for all samples extracted by the Wagner CTAB protocol, even after an additional purification step (Table 2). This may indicate the presence of potential contaminants which absorb at 230 nm [26].

The differences observed in DNA quality is probably due to the major differences between the tow protocols used : the adapted CTAB protocol is successfully optimized and upgraded by including enzymatic digestion using proteinase K and RNase, the addition of the CTAB precipitation solution that remove additional possible polysaccharides and polyphenolic compound that might be combined with DNA and therefore affecting the purity and quality of the extracted DNAs, the incubation steps involved in this protocol to improve the DNA quality,and the addition of an overnight isopropanol precipitation step, also the ultracentrifugation speed and incubation time used can explain also the differences in quality and purity obtained.

DNAs extracted with Wagner CTAB protocol were of bad quality, these maybe indicate the presence of polysaccharides and polyphenolic compound, the presence of such kind of contaminant may interfere negatively during downstream reactions like PCR amplification or DNA digestion [14]. To test this hypothesis, we decided to amplify the coding region of the THC synthase gene by PCR using DNA extracted by both protocols as template.

PCR amplification of THC synthase gene from different samples

The quality and integrity of DNA obtained by the two protocols was assessed by PCR amplification of full-length coding region of the THCA synthase gene using specific primers (a/b).

The PCR amplification of THCA synthase was successful on DNA obtained by the Adapted CTAB protocol (Figure 2A). However, no amplification was obtained with DNA extracted by the Wagner protocol (Figure 2B1). This may be explained by the presence of PCR inhibitor in DNA extracted by the Wagner CTAB protocol, and/or by the loss of DNA integrity.

Electrophoretic analysis of genomic DNA extracts

DNA samples extracted with Wagner CTAB protocol showed smearing bands, which indicate that the DNAs were highly degraded and/or fragmented and may explain the PCR failure of THC synthase gene. (Figure 3B).

DNA samples extracted with the Adapted CTAB protocol show one predominant band in all samples (Figure 3A) indicating intact gDNA, and thus improved preservation of DNA integrity making it possible to perform amplification across the sequence of the gene of interest. This amplified DNA was further submitted to another downstream method test based on DNA sequencing.

Sequencing

To assess the possibility of sequencing amplified DNAs, we have chosen to sequence the complete coding region of the THCA synthase gene in one sample (Sample1). Amplicons obtained by the combination of primers (mentioned above) that cover the total sequence were submitted to sequence analysis on the ABI genetic analyser. All electropherograms were of good quality. After the assembly of all sequence data, a complete sequence of the THCA synthase gene was generated.

These results demonstrate that this protocol was proven to be suitable for the extraction of high quality DNA from cannabis resin (Hashish) that could be used as a template in routine DNA sequencing techniques.

Conclusion

Our study demonstrates that the Adapted CTAB protocol for difficult matrix allowed us to obtain a high quality DNA from cannabis resin samples. This DNA quality was sufficient to perform amplification of a specific gene using PCR technique. Wagner CTAB protocol is largely used to extract genomic DNA from different plants, but it is not ideal in the case of cannabis resin. In our experiments, the adapted protocol was used in routine extraction of DNA from cannabis resin. The high DNA quality obtained permit downstream DNA analysis techniques such as PCR and sequencing. These techniques will contribute to the identification of the geographic origin of seized cannabis resin (Hashish) conducted by the police or by Customs in checkpoints and therefore establish the connection traffic pathways.

Hence the necessity of finding a reliable protocol for the extraction of highly purified nucleic acids derived from Cannabis sativa resin seizure which is the most common form of seizure in checkpoints.

Developing such kind of DNA extraction from unusual material derived from cannabis plant (like Hashish) will be of great interest given its close association with forensic casework.

Our sincere thanks for the support of the Functional Genomic Platform; (UATRS) at the Moroccan Centre for Scientific and Technical Research (CNRST).

This work was supported by the Functional Genomic Platform; (UATRS) at the Moroccan Centre for Scientific and Technical Research (CNRST). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.