WO1998024929A1

WO1998024929A1 - Detection of cannabis by dna

Info

Publication number: WO1998024929A1
Application number: PCT/GB1997/003320
Authority: WO
Inventors: Adrian Matthew Thornton Linacre; James William Thorpe
Original assignee: University Of Strathclyde
Priority date: 1996-12-03
Filing date: 1997-12-03
Publication date: 1998-06-11
Also published as: EP0941367A1; AU5231098A; GB9625086D0; CA2274320A1

Abstract

The present invention relates to the use of a cannabis specific nucleotide sequence for the detection of cannabis material, in particular the use of a cannabis specific DNA sequence. Detection of cannabis material may be determined by utilising the cannabis specific nucleotide sequence as a primer in polymerase chain reaction (PCR) or sequencing studies, or as a probe in hybridisation studies.

Description

DETECTION OF CANNABIS BY DNA

The present invention relates to the use of a cannabis specific nucleotide sequence for the detection of cannabis material, in particular the use of a cannabis specific DNA sequence.

Drug trafficking is a major problem worldwide and there is considerable investment by Customs and Excise and other law enforcement agencies to prevent the import and supply of controlled substances e.g. drugs. When samples of drugs are found, either as small quantities or as bulk supplies, it is useful to link them to larger batches to assist in identifying the supply routes. Information on distribution and supply routes for such drugs can lead to their interception at comparatively few sites in the country of origin rather than at the numerous street outlets. This is considered to be more effective law enforcement.

There are currently a series of tests that can indicate the presence of a controlled drug. These presumptive tests include microscopy, chemical spot tests and Thin Layer Chromatography. On the basis of a positive result from these tests, techniques such as Gas Chromatography Mass Spectrosmetry or High Pressure Liquid Chromatography are used to identify the drug. Further examination using gas and liquid chromatography can be used to compare samples but the composition of the samples does vary with time which reduces the value of this approach. Moreover, such techniques employ expensive and complex equipment which are not suitable for routine analysis.

It has been envisaged that the same techniques used by the forensic scientist for human DNA profiling could be applied to any biological material that contains DNA. While most forensic analysis is aimed at human individualisation, application of DNA profiling could be used for the examination of other species.

If DNA profiling is to be applied to cannabis for its unambiguous identification and to give an indication of its geographical origin then a DNA profiling technique which examines areas of the cannabis genome displaying a degree of intra-species variation and a high degree of inter- species variation is preferable. Work with Random Amplification of Polymorphic DNA (RAPD) has been performed previously on Cannabis sativa (Gillian et al . 95). However, problems with specificity and reproducibility have prevented RAPD being adopted as a reliable technique.

It is an object of the present invention to provide an improved method of identifying cannabis material in a sample using a cannabis specific nucleotide sequence.

In all land plants and algae analysed to date chloroplast DNA (cpDNA) has been found to exist as a single circular molecule ranging in size from 83-292 kb (Bohnert et al . 82). The chloroplast contains many well characterised genes such as ribose-1, 5-bisphosphate carboxylase (rjcL) which has allowed the study of cpDNA sequence polymorphisms and these are now widely used to investigate inter-species relationships (Clegg 93) . Due to the relatively low rate of evolutionary change cpDNA has been little used for intra-species variations but the use of Restriction Fragment Length Polymorphism (RFLP) on cpDNA has indicated that intra-species variation does exist.

The polymerase chain reaction (PCR) (Saiki et al . 1988) can be used to specifically amplify fragments of DNA using primers which are based upon known DNA sequences. Conserved priming sites which flank three chloroplast tRNA genes ( trn , trnF, and trnT) have been identified from which universal amplification primers have been designed (Taberlet et al . 93). Such universal primers have been used on a wide range of species and previous work using these primers has successfully amplified cpDNA fragments from such diverse plants as mosses, ferns and higher dicotyledons (Fanagan et al . 94), but to date no studies on cpDNA of cannabis are known to have been carried out. Additionally, the intron within the tr.nL gene and the intergenic spacer between the 3 ' exon and the trnF gene have been shown to exhibit variation in both length and sequence (Fanagan et al . 94), which may be used in inter- species identification.

The present invention has identified polynucleotide sequences within the Cannabis sativa chloroplast genome which are cannabis specific. Such cannabis specific polynucleotide sequences may be used to identify cannabis material in samples and may be included in a definitive test for cannabis material. One aspect of the present invention provides isolated polynucleotide sequence (s) unique to Cannabis sativa plants. The sequence(s) may be RNA or DNA sequence(s). The sequence (s) may be found within the Cannabis sativa chloroplast genome, more particularly within the Cannabis sativa chloroplast genome situated between and within the tr.nL 5'exon and the trnF gene. In principle any Cannabis sativa specific polynucleotide sequence (s) from the above identified region of the Cannabis sativa chloroplast genome may be used in polymerase chain reaction (PCR) studies, hybridisation studies, or sequencing studies and may for example be derived from Cannabis sativa specific or fragments of the sequences shown in Figure 1.

The invention still further provides nucleotide sequence (s) which is/are similar to the above disclosed DNA sequences. By "similar" is meant a sequence which is capable of hybridising to a sequence which is complementary to the inventive nucleotide sequence. The present invention also provides anti-sense or complementary nucleotide sequence (s) which is/are capable of hybridising to the inventive nucleotide sequence. It will be appreciated that such anti-sense or complementary nucleotide sequence(s) need not be identical in sequence to a complementary sequence of the inventive sequence, but that the anti-sense or complementary sequence (s) must be capable of hybridising to the inventive sequence. If a Cannabis sativa specific polynucleotide sequence is to be used as a primer in PCR or sequencing studies, the polynucleotide must be capable of hybridising to Cannabis sativa nucleic acid and capable of initiating chain extension from the 3' end of the polynucleotide, but not able to correctly initiate chain extension from non Cannabis sativa sequences.

If a Cannabis sativa specific test polynucleotide sequence is to be used in hybridisation studies, to test for the presence of Cannabis sativa nucleic acid in a sample, the test polynucleotide should preferably remain hybridised to a sample polynucleotide under stringent conditions, with either the test or sample polynucleotide preferably being supported. Generally the test polynucleotide sequence is at least 50 bases in length, and may be labelled by suitable random priming techniques known in the art. Preferably the test polynucleotide sequence is at least 200 bases in length and may even be several kilobases in length. Thus, either a denatured sample or test sequence is preferably first bound to a support and hybridization is effected for a specified period of time generally at a temperature of between 50 and 70 °C in double strength SSC (2xNaCl 17.5g/l and sodium citrate (SC) at

8.8g/l) buffered saline containing 0.1% sodium dodecyl sulphate (SDS) followed by rinsing of the support at the same temperature but with a buffer having a reduced SSC concentration. Depending upon the degree of stringency required, and thus the degree of similarity of the sequences, such reduced concentration buffers are typically single strength SSC containing 0.1%SDS, half strength SSC containing 0.1%SDS and one tenth strength SSC containing 0.1%SDS. Sequences having the greatest degree of similarity are those the hybridization of which is least affected by washing in buffers of reduced concentration. It is most preferred that the sample and inventive sequences are so similar that the hybridization between them is substantially unaffected by washing or incubation in one tenth strength sodium citrate buffer containing 0.1%SDS.

It will be immediately evident to those skilled in the art that the Cannabis sativa specific polynucleotide sequence (s) of the present invention may be utilised in so- called gene-chip technology in order to provide a gene- chip, which may be used in Cannabis sativa nucleic acid detection.

Cannabis sativa specific oligonucleotides may be designed to specifically hybridise to Cannabis sativa DNA. They may be synthesised, by known techniques and used as primers in PCR or sequencing reactions or as probes in hybridisations designed to detect the presence of Cannabis sativa material in a sample. The oligonucleotides may be labelled by suitable labels known in the art, such as, radioactive labels, chemiluminescent labels or fluorescent labels and the like. Thus, the present invention also provides Cannabis sativa specific oligonucleotide probes and primers.

The term "oligonucleotide" is not meant to indicate any particular length of sequence and encompasses nucleotides of between 10b to lkb in length, more preferably 12b-500b in length and most preferably 15b to 100b. Examples of 10 oligonucleotides according to the present invention which are suitable for use in hybridisation, sequencing and/or PCR studies are:

TM

Primer I 5i '' -GAGGTTTCTAATTTGTTATGTT-3 ' 51°C

Primer II 5i '' -ACTAGAGGACTTGGACTATGTC-3 ' 59 °C

Primer III 5>''-TCCGGTTTTCTGAAAACAAACAAG-3' 61°C

Primer IV 5I '' -TTGGCTGCGTTAATCCGGATTTCT-3 ' 65 °C

Primer V 5 '' -TTGATTTTTCATGAAAAATCAAAG-3 ' 53 °C

Primer VI 5i '' -AATCTGATAGATTTTTTGAAGACT-3 ' 55 °C

Primer VII 5i''-GGTTCAAGTCCCTCTATCCCCAAA-3' 67 °C

Primer VIII 5i '' -TTATTTATCCTCTCATTCCTTAGA-3 ' 55 °C

Primer IX 5i '' -ATGTTTCTCGTTCATTCTAACTTA-3 ' 59°C

Primer X 5I '' -GAATGACCTTTTTTTTATTATCAG-3 ' 55°C (melting temperatures (Tm) are calculated according to the formula of; 2°C for every A or T, plus 4°C for every G or C, minus 5°C)

The above oligonucleotides are derived from the Cannabis sativa sequences shown in Figure 1 and may be manufactured according to known techniques. Figure 2 shows in detail where Primers I to X above would anneal or hybridise to a consensus of the Cannabis sativa sequences shown in Figure 1. Experimental evidence supporting the specificity of Primers I and II and their use in identifying Cannabis sativa material is described in detail later herein. It will be appreciated to one skilled in the art that the primers are shown in one 5 '-3' orientation, but that the complementary sequence of each primer may also be used where appropriate. Thus, for example, primer IV (5'-TTGGCTGCGTTAATCCGGATTTCT-3') and the complementary primer to primer IV (i.e. 5'-AGAAATCCGGATTAACGCAGCCAA-3 ' ) are encompassed by the scope of the present invention.

To the present inventors knowledge, none of the above oligonucleotides show identical homology to any previously sequenced plant nucleic acid, as determined by DNA database analysis, at the date of filing. Additionally, the above oligonucleotides show less than 60% identity with previously sequenced chloroplast nucleic acid from other plant species (i.e. not Cannabis sativa) .

Oligonucleotides which are generally greater than 30 bases in length should preferably remain hybridised to a sample polynucleotide under the stringent conditions mentioned above. Oligonucleotides which are generally less than 30 bases in length should also preferably remain hybridised to a sample polynucleotide but under different conditions of high stringency. Typically the melting temperature of an oligonucleotide less than 30 bases may be calculated according to the formula of; 2°C for every A or T, plus 4°C for every G or C, minus 5°C. Hybridisation may take place at or around the calculated melting temperature for any particular oligonucleotide, in 6 x SSC and 1% SDS. Non specifically hybridised oligonucleotides may then be removed by stringent washing, for example in 3 x SSC and 0.1% SDS at the same temperature. Only substantially identically matched sequences remain hybridised i.e. said oligonucleotide and corresponding Cannabis sativa nucleic acid.

When oligonucleotides of generally less than 30 bases in length are used in sequencing and/or PCR studies, the melting temperature may be calculated in the same manner as described above. The oligonucleotide may then be allowed to anneal or hybridise at a temperature around the oligonucleotide's calculated melting temperature. In the case of PCR studies the annealing temperature should be around the lower of the calculated melting temperatures for the two priming oligonucleotides. It is to be appreciated that the conditions and melting temperature calculations are provided by way of example only and are not intended to be limiting. It is possible through the experience of the experimenter to vary the conditions of hybridisation and thus anneal/hybridise oligonucleotides at temperatures above their calculated melting temperature. Indeed this can be desirable in preventing so-called non-specific hybridisation from occurring.

It is possible when conducting PCR studies to predict an expected size of PCR product obtainable using an appropriate combination of two of the above described oligonucleotides, based on where they would hybridise to the sequence in Figure 1. If, on conducting such a PCR on a sample of chloroplast DNA, a fragment of the predicted size is obtained, then this is predictive that the DNA is Cannabis sativa . Furthermore, amplification of Cannabis sativa chloroplast DNA using PCR techniques may generate products which differ in size from products obtained from any other plant species so far determined. Such a size difference may constitute a presumptive test for the presence of Cannabis sativa .

It has been previously reported (Gillian et al., 1995) that DNA can be extracted from cannabis resin and PCR amplification may be carried out on such DNA to confirm for the presence of Cajxnajbis sativa .

Oligonucleotides corresponding to intra-species variable portions of the above identified region could be used for intra-species identification using PCR.

The present invention also encompasses cannabis detection kits including at least one oligonucleotide which is Cannabis sativa specific, as well as any necessary reaction reagents, washing reagents, detection reagents, signal producing agents and the like for use in the test formats outlined above.

In a further aspect there is also provided use of a Cannabis sativa specific polynucleotide in the detection of Cannabis sativa in a sample.

In a yet further aspect there is provided use of a Cannabis sativa specific polynucleotide in a PCR for the detection of Cannabis sativa in a sample.

Examples of the present invention will now be described by way of example only. All Techniques were carried out according to Sambrook et al. (1989) unless otherwise indicated. The Examples will also be better understood with reference to the drawings, in which: Figure 1 shows the DNA sequence and alignment of a portion of the chloroplast genome of four Cannabis sativa stocks; Figure 2 shows a consensus sequence of the sequences shown in Figure 1 and identified primer binding sites; Figure 3 shows a sequence alignment. The sequence of Cannabis clone 22 is shown on the bottom line. Where there is a dot (no sequence) there is no homologous sequence. Homologous sequences are where the same sequence in the Cannabis clone is directly underneath an identical base in one or more of the above sequences. When a plant species is shown more than once the additional sequences are the DNA sequences between trnL and trnF genes found elsewhere in the genome;

Figure 4 shows the amplification with Cannabis sativa specific primers on C. sativa . The PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and visualised under u.v. Lane A was the 100 bp ladder, lane B amplification products with primers b and c, lane C amplification products with I and II, and lane D amplification products with b, c, I, and II. Figure 5 shows the amplification with Cannabis sativa specific primers on C. sativa . Lane A shows a 100 bp ladder; lane B shows the amplification product using universal primers a and d; lane C shows the amplification product using primers I and II; and lane D shows the amplification products with primers a, d, I and II. 11a Figure 6 shows the amplification products with universal primers and C. sativa primers from numerous botanic specimens. The PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide and visualised under u.v. Lane A shows the PCR product using primers b,c, I and II from arabadopsis; Lane B, of bean using b,c,I and II; Lane C, of bean using b and c; lane D, of beansprout using b,c,I and II; lane E, of cabbage using b,c,I and II; lane F, of corn using b,c,I and II; lane G of corn using b and c; lane H, of green pepper using b,c,I and II; lane I lOObp ladder; lane J, of rice using b,c,I and II; lane K, of grass using b,c,I and II; lane L, of grass using b and c; lane M of kidney bean using b,c,I and II; lane N, of poppy using b,c,I and II; lane 0, of tobacco using b,c,I and II; lane P, of tomato using b,c,I and II and lane Q, of wheat using b,c,I and II.

Example 1

Amplification and Sequencing of Cannabis Chloroplast DNA with universal Primers

DNA Extraction

One leaf from each of four Cannabis sativa plants was obtained. One leaf was from a plant of Indian origin and the other three leaves were from plants of South African origin. Each leaf was dried and 500mg of plant material was removed. The leaf material was ground in a mortar and pestle with the addition of liquid nitrogen. The powdered lib leaf material was transferred to a 1.5ml Eppendorf tube to which 400μl of lysis buffer (50mM Tris-HCl, 20mM EDTA, 1% SDS, NaOH to pH 8.0) was added. To this lOμl of lOO g/ml proteinase K was added and the solution incubated at 56°C for 30 minutes. After this period 125μl of 5M potassium acetate was added, the tube mixed at room temperature for 5 minutes and then incubated at 65 °C for 20 minutes. To this 700μl of chloroform (stored at -20°C) and I20μl of Boric-PSG slurry (lg Boric-PSG in 1.2ml lxTE pH7.4) (Scotlab Limited Glasgow, Scotland) was added. The tube was placed on a tilt shaker for 10 minutes after which it was centrifuged at 2,500 rpm for 5 minutes. The upper aqueous layer was transferred to a new tube, chloroform extracted, and the centrifuge step repeated. The upper layer was transferred to a 2ml tube to which 2 volumes of

12 ice cold ethanol was added to precipitate the DNA. After mixing the tube it was spun at 14,000 rp for 3 minutes. The supernatant was discarded and the remaining DNA pellet dried, and finally resuspended in 50μl of TE. The same method was used for all DNA extractions from the other plant species tested. In each case the same weight of starting material was used.

PCR Amplification of Cannabis Sativa DNA

Positions and directions of the universal primers used to amplify the non-coding regions of cpDNA in Cannabis sativa are shown below. The points of the arrows indicate the direction of amplification. Primer a is situated within the trnL 5'exon, primer b (and primer of which is the complement of primer b) within the trnL 3 ' exon and primer c within the trnF gene.

trnL trnL trnF

5'exon

a→ b→ *-c ^d

Sequences of the universal primers used in the initial amplification of Cannabis sativa chloroplast DNA (cpDNA) are shown below: 13

Primer Name Sequence 5' - 3' a CGAAATCGGTAGACGCTACG b GGTTCAAGTCCCTCTATCC c ATTTGAACTGGTGACACGAG d GGATAGAGGGACTTGAACC

PCR Amplification with Universal Primers a and c

PCR amplifications were performed in a total volume of 50μl. Each reaction contained: 20ng of Cannabis sativa DNA, 200μM of each deoxyribonucleotide triphosphate (dNTPs) , PCR buffer (lO M Tris HC1 pH8.3 , 50mM KCl, 1.2mM MgCl₂) , 2 units Taq polymerase (Dynazyme, FMC) , and lOOpM of each primer. The reaction solution was overlaid with two drops of mineral oil. Amplification proceeded for 35 cycles at 94 °C for 1 minute, 54 °C for 1 minute and 72 °C for 1 minute followed by 72 °C for 7 minutes, using a hybaid thermacycler instrument. The PCR products were electrophoresed on a 3% agarose gel and detected by staining with ethidium bromide and photographed with Polaroid 667 film. The four cannabis sativa plants all produced the same size fragment of approximately 800 bp when visualised.

Cloning and Sequencing of PCR Products

PCR products from the amplification with primers a and c from Cannabis sativa leaf extracts were cloned into the plasmid vector pCRII as part of the TA Cloning kit (Invitrogen) . In each cloning reaction 20ng of the PCR product were ligated into 50ng of the TA vector (pCRII) . The ligations were otherwise as according to the 14 manufacturers instruction. The transformations were performed using the commercially supplied E . coli cells of the TA cloning kit. White colonies were picked from each transformation and mini-prepped using the Sigma Plasmid Pure Kit. The presence of a PCR insert was confirmed by restriction digest analysis with the enzyme Eco RI. Automated DNA sequencing was performed for each of the recombinant plasmids using an Applied Biosystems 373A sequencer. The Dye Terminator method of sequencing was used throughout using PRISM Taq Cycle sequencing kits (Applied Biosystems, Warrington, UK) , according to manufacturers instructions.

Alignment and Statistical Analysis

The Cannabis sativa DNA sequences were aligned by the computer programme SeqEd™ (Applied Biosystems, Warrington, UK) . Alignment of the Cannabis sativa sequences obtained from the four Cannabis sativa plants was performed by PILEUP (Smithies et al. 1981 and Feng & Doolittle 1987). The sequences were then compared to the EMBL database using the programme FastA (Lipman & Pearson 85) .

A comparison of the four cannabis sequences (denoted 7, 20, 22, 24) is illustrated in Figure 1. There is a homology of 99.8% between the two least homologous sequences (7 and 24) illustrating that there exists little intraspecies sequence diversity. However, differences between the four sequences can be observed. Such differences may be exploited when designing an intra 15 species Cannabis sativa test. Oligonucleotides which are specific to one plant may be designed which may be used in tests for identifying Cannabis sativa plants from a particular geographical location.

Comparison of Cannabis sativa tRNA Sequence to all known tRNA Chloroplast Sequences

The full 818 bases of the sequence from the Cannabis sativa plant 22 were compared to other DNA sequences using the EMBL DNA database. The results of this search, only showing the 32 closest matching plants, are illustrated in Figure 3. Almost all the sequences shown in Figure 3 are from the tRNA gene of the chloroplasts of different plants. The other sequences are from tRNA genes located within nuclear DNA.

Figure 2 shows the sequenced Cannabis sativa DNA to have significant differences, throughout the 818 bases, when compared to the most closely related correponding DNA sequences from other plant species.

Example 2

Specific Amplification of Cannabis sativa DNA

Design of Cannabis Specific Primers

From the Cannabis sativa DNA shown in Figure l, priming sites that could be used in a PCR amplification and would be specific to Cannabis sativa and no other known plant were designed (see Figure 2) . DNA sequences suitable for use as primers in amplification reactions were designed 16 by selecting DNA sequences of between 20 and 25 bases in length and comparing the sequence to the DNA Database using the FastA method of the GCG Wisconsin sequencing package (Higgins & Sharp 1989, and Lip an & Pearson 1985) . Those DNA sequences with a high degree of homology to known nucleic acid sequences were discarded. Only sequences which showed a low degree of homology to known nucleic acid sequences were analysed further. Ten possible primers (I to X) were identified and the sequences are described hereinbefore. Two of the 10 primers (primers I and II) have been used in PCR amplification studies. FastA analysis revealed a maximum homology with other plant species of 54% for primer I and 50% for primer II and most importantly no homology at the 3' end of each primer.

Experimental conditions: PCR Amplification with Universal Primers b, c, and Cannabis Sativa Specific Primers I and II PCR amplifications were performed in a total volume of 50μl. Each reaction contained: 20ng of plant DNA, 200μM of each deoxyribonucleotide triphosphate (dNTPs) , PARR Excellence PCR buffer (Cambio Limited, Cambridge, UK) , 2 units AmpliTaq Gold polymerase (PE Applied Biosystems, Warrington UK) , and lOOpM of each relevant primer used. Amplification proceeded for 35 cycles of 94 °C for 30 seconds, 60°C for 30 seconds and 72 °C for 30 seconds followed by 72 °C for 7 minutes using a Perkin Elmer 2400 thermalcycler. The PCR products were electrophoresed on a 2% agarose gel and the gels were stained with ethidium 17 bromide and photographed with Polaroid 667 film.

Amplification of Cannabis sativa DNA

The Cannabis sativa specific primers (I and II) were used in a PCR amplification using DNA extracts from

Cannabis sativa and PCR products of the expected sizes (199 bp) were detected (see lane C of Figure 3) . This is considerably smaller than that produced using primers a and c (818 bp) , the sequence of which is shown in Figure 1.

The proposed Cannabis specific primers also lie inside the

DNA sequences b and c (see previously) . The amplification product using b and c from the cannabis clones produced a fragment size of 349 bp as predicted (see lane B of Figure

4) . The Cannabis sativa specific primers (I and II) were used in conjunction with the universal chloroplast primers b and c. This duplex test when conducted using Cannabis sativa should produce two PCR products, a PCR product of previously known size for b and c (361 bp) and a further product using primers I and II (199 bp) . A product of the size amplified using primers b and c was produced but the second main product (265 bp) was determined to be an intermediate product produced from primers b and II (see lane D of Figure 4) . This intermediate product is produced due to the preference of primer b to bind rather than primer I under the annealing conditions performed in the

PCR. DNA extracted from other plants should produce a product consistent with that produced with the primers b and c but no other product, as primers I and II should only 18 produce an amplification product if the I and II priming sites are present, i.e. that the material to be amplified is Cannabis sativa material.

In order to design a test which would not produce an intermediate product as produced from primers b and II, a further universal primer d was used. Primer d is in fact the complement of primer b. A control reaction using universal chloroplast primers a and d gives a product of size 471 bp, which is easily discernible from the 199bp product obtained using cannabis specific primers I and II. See figure 5 which shows the amplification using primers a and d producing the 471 bp fragment; an amplification using cannabis specific primers producing the 199 bp fragment; and an amplification reaction with both the universal and the cannabis specific primers producing the 471 bp and 199 bp fragment sizes.

Amplification with universal chloroplast primers and Cannabis sativa Specific Primers on Various Plant Species

Duplex PCR amplification on a range of plant DNA samples with the primers b, c, I, and II were performed and the resulting products were separated on an agarose gel.

The plants were chosen for the amplification reactions either by the close homology as shown in Figure 3 or due to their close taxonomic association. The results of these amplifications are shown in Figure 6. Reactions with only a single set of primers were also performed when the fragment size produced in the duplex reaction could not be unambiguously assigned to one set of primers. 19 Amplification from all the plants produced PCR products that were consistent with the amplification using the b and c primers only. No other PCR product was detectable, indicating that the I and II primers were not priming on any part of their genome.

Experimental testing of volunteers hands after handling Cannabis sativa material

Volunteers were used who could verify that they had not knowingly been in contact with cannabis before. They were asked to wash their hands thoroughly in soap and water before any test began. The area of the hand tessted was the index finger and thumb as this is the area of the hand most likely to be used in preparing cannabis preparations such as 'joints'.

As a negative control a cotton wool swab wass taken from the index finer and thumb immediately before the addition of any cannabis. The volunteer was then asked to was their hands again. The volunteer was then asked to handle C. sativa vegetative material between the index finger and thumb. The time period of handling was measured in seconds and was such that no vegetative material could be visualized on the hands. The index finger and thumb were then swabbed with a cotton wool bud which had previously been soaked in sterile water.

The cotton wool bud was then removed to a sterile 1.5ml tube containing 1 ml of extraction buffer (50mM Tris- HCl, pHδ.l, 20mM EDTA, pH8.0, 0.2% Bovine serum albumin, 1% polyvinylpyrrolidone, 0.1% 2-mercatoethanol) . The base of 20 the tube was then placed inside another sterile 1.5ml tube and centrifuged for 10 minutes until the fluid had been remove from the cotton wool. Sodium dodecyl sulphate (SDS) was added to the solution to a final concentration of 2%. The sample was incubated at 65°C for 15 minutes after which it was placed on ice. To the solution, 285 μl of 5M potassium acetate was added and the tube incubated for 15 minutes on ice. The tube was then spun at 11,000 g after which the solution was removed leaving any precipitated protein behind. This supernatant was removed into a 10,000 MW Vivaspin concentrator (Greiner Labs. U.K.) and centrifuged at ll,000g for 20 minutes. The solution containing any C. sativa DNA was now concentrated to a volume of 20 μl.

The negative controls wee treated in exactly the same manner .

Amplifications by the polymerase chain reaction (PCR) were preformed as previously described (see Example 1) . Amplifications contained the universal primers a and d and the Cannabis specific primers I and II. The products produced by the PCR were separate on agarose gels and visualized as previously described. The negative controls produced one discernible product. PCT samples originating from the hand swabs taken from volunteers who had handled C. sativa produced PCR products of sizes corresponding to those anticipated with a and d and I and II amplify C. sativa DNA (see for Example Figure 7) . 21 The same experiment was repeated for 5 further individuals who all gave the same results, ie one discernible product for the negative control and two products from the test, after the individuals had handled cannabis material.

Thus, on the basis of this work, it can be seen that Cannabis sativa specific primer (s) may be used to identify whether or not an unknown sample contains Cannabis sativa nucleic acid and consequently comprises Cannabis material. In principle any single primer or suitable combination of primers from priners I to X or complementary primers thereof may be used to identify cannabis material. Amplification using primers I and II of a product of approximately 199 bases in length indicates that Cannabis sativa nucleic acid is present in a test sample. Amplification of a product of different size, or no amplification at all, signifies that Cannabis sativa nucleic acid and therefore Cannabis material is not present in the sample.

Universal primers, such as, primers b and c or a and d may be used as an optional positive control to ensure that nucleic acid has been correctly extracted from the sample and that the PCR conditions are suitable for amplification to occur. 22 References

1. R. Gillan et al . Science & Justice 35: 169-177 (1995).

2. H.J. Bohnert, E.J. Crouse & J.M. Schmitt Encyclopaedia of Plant Physiology 143 (1982).

3. M. Clegg, Proc. Natl. Acad. Sci. USA 90: 363-367

(1993) .

4. R.K. Saiki et al . Science 239: 487-491 (1988).

5. P. Taberlet, L. Gielly, G. Pautou & J. Bouvet, Plant Molecular Biology 17: 1105-1109 (1991).

6. B. Fanagan et al . BioTechniques 16: 484-494 (1994).

7. Sambrook, J. , Fritsch, E.F., and Maniatis, T. (1989) In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, Cold Spring Harbour New York.

8. 0. Smithies et al. Cell 26: 345-353 (1981).

9. D.F. Feng, & R.F. Doolittle, Journal of Molecular Evolution 25: 351-360 (1987).

10. D.J. Lipman & W.R. Pearson, Science 227: 1435-1441 (1985) . 23 11. D.G. Higgins & P.M. Sharp Computer Applications in the Bioscience 5: 151-153 (1987) .

Claims

24 CLAIMS

1. An isolated polynucleotide sequence (s) unique to Cannabis sativa plants.

2. An isolated polynucleotide sequence (s) according to claim 1 wherein the sequence (s) is/are found with the Cannabis sativa chloroplast genome.

3. An isolated polynucleotide sequence (s) according to claim 2 wherein the sequence is situated between and within the trnL 5'exon and the trnF gene.

4. An isolated polynucleotide sequence (s) according to claim 3 wherein the sequence is substantially the sequence shown in Figure 1, or a Cannabis specific polynucleotide fragment or fragments thereof.

5. An isolated polynucleotide fragment or fragments according to claim 4 selected from the group:

Primer I 5' -GAGGTTTCTAATTTGTTATGTT-3 ' ;

Primer II 5' -ACTAGAGGACTTGGACTATGTC-3 ' ;

Primer III 5' -TCCGGTTTTCTGAAAACAAACAAG-3 '

Primer IV 5' -TTGGCTGCGTTAATCCGGATTTCT-3 '

Primer V 5' -TTGATTTTTCATGAAAAATCAAAG-3 '

Primer VI 5' -AATCTGATAGATTTTTTGAAGACT-3 '

Primer VII 5' -GGTTCAAGTCCCTCTATCCCCAAA-3 '

Primer VIII 5' -TTATTTATCCTCTCATTCCTTAGA-3 ' 25 Primer IX 5'-ATGTTTCTCGTTCATTCTAACTTA-3 ' ; and Primer X 5'-GAATGACCTTTTTTTTATTATCAG-3 ' .

6. An isolated polynucleotide sequence (s) or fragment (s) according to any proceeding claim wherein the sequence is RNA or DNA.

7. An isolated polynucleotide sequence (s) or fragment (s) which is anti-sense or complementary to a sequence (s) or fragment (s) according to any preceding claim.

8. An isolated polynucleotide sequence (s) according to any preceding claim wherein the sequence (s) is/are between 10b and lkb in length.

9. An isolated polynucleotide sequence (s) according to any preceding claim for use as a probe in hybridisation studies wherein the probe remains hybridised to a sample polynucleotide under stringent conditions.

10. An isolated polynucleotide sequence (s) according to claim 9 wherein the polynucleotide sequence (s) is greater than about 30 bases in length and remains hybridised to the sample in tenth strength SSC containing 0.1% SDS at a temperature of 70°C or greater. 26

11. An isolated polynucleotide sequence (s) according to claim 9 wherein the polynucleotide sequence (s) is less than about 30 bases in length and remains hybridised to the sample at or around the calculated melting temperature for the polynucleotide sequence (s) in 3 x SSC and 0.1% SDS.

12. An isolated polynucleotide sequence (s) according to any one of claims 1 to 8 for use as a primer in PCR or polynucleotide sequencing studies, wherein the primer is capable of hybridising to Cannabis sativa nucleic acid and capable of initiating chain extension from the 3' end of the polynucleotide, but not able to correctly initiate chain extension from non Cannabis sativa polynucleotide sequences.

13. Use of a Cannabis Sativa specific polynucleotide (s) according to any proceeding claim in the detection of Cannabis sativa in a sample.

14. Use of a Cannabis sativa specific polynucleotide (s) according to claim 13 in a hybridisation study for the detection of Cannabis sativa in the sample.

15. Use of a Cannabis sativa specific polynucleotide (s) according to claim 13 in a PCR for the detection of Cannabis sativa in the sample. 27

16. Use of a Cannabis sativa specific polynucleotide (s) according to claim 15 to generate a product from Cannabis sativa vegetative material whilst not producing a product, or a product of different size from vegetative material from other sources.

17. Use of a Cannabis sativa specific polynucleotide (s) according to claim 16 together with a further non- Cannabis sativa specific polynucleotide (s) as a control.

18. A kit for use in detection of Cannabis sativa comprising a polynucleotide (s) according to any one of claims 1 to 12.