Elsevier

Legal Medicine

Volume 26, May 2017, Pages 33-40
Legal Medicine

Short Communication
Developmental and internal validation of a novel 13 loci STR multiplex method for Cannabis sativa DNA profiling

https://doi.org/10.1016/j.legalmed.2017.03.001Get rights and content

Highlights

  • A novel 13 loci cannabis STR multiplex method was developed and validated.

  • Six new cannabis STR tetranucleotide markers were included.

  • A sequenced allelic ladder was designed to genotype 101 C. sativa samples.

  • The sensitivity is 0.13 ng DNA and the power of discrimination is 1 in 55 million.

  • Well-balanced heterozygous peaks with minimal artifacts and stutter were observed.

Abstract

Marijuana (Cannabis sativa L.) is a plant cultivated and trafficked worldwide as a source of fiber (hemp), medicine, and intoxicant. The development of a validated method using molecular techniques such as short tandem repeats (STRs) could serve as an intelligence tool to link multiple cases by means of genetic individualization or association of cannabis samples. For this purpose, a 13 loci STR multiplex method was developed, optimized, and validated according to relevant ISFG and SWGDAM guidelines. The STR multiplex consists of 13 previously described C. sativa STR loci: ANUCS501, 9269, 4910, 5159, ANUCS305, 9043, B05, 1528, 3735, CS1, D02, C11, and H06. A sequenced allelic ladder consisting of 56 alleles was designed to accurately genotype 101 C. sativa samples from three seizures provided by a U.S. Customs and Border Protection crime lab. Using an optimal range of DNA (0.5–1.0 ng), validation studies revealed well-balanced electropherograms (inter-locus balance range: 0.500–1.296), relatively balanced heterozygous peaks (mean peak height ratio of 0.83 across all loci) with minimal artifacts and stutter ratio (mean stutter of 0.021 across all loci). This multi-locus system is relatively sensitive (0.13 ng of template DNA) with a combined power of discrimination of 1 in 55 million. The 13 STR panel was found to be species specific for C. sativa; however, non-specific peaks were produced with Humulus lupulus. The results of this research demonstrate the robustness and applicability of this 13 loci STR system for forensic DNA profiling of marijuana samples.

Introduction

Forensic DNA typing is typically performed on human DNA samples. However, the molecular analysis of plant DNA is increasingly being studied and considered for use in criminal justice systems around the world [1], [2], [3]. In the field of forensic plant science, plant DNA can be used to link a suspect or a victim to a location (crime scene) or in the case of marijuana, can be used to aid in the investigation of drug cases. In the United States, marijuana is the most commonly used illicit controlled substance [4]. Consequently, it is a highly trafficked drug to and within the United States by organized crime syndicates. The development of a validated method using molecular techniques such as short tandem repeats (STRs) for the genetic identification of C. sativa may aid in the individualization and origin determination of cannabis samples as well as serving as an intelligence tool to link cannabis cases (e.g., illegal traffic at the US-Mexico border).

In 2003, the first polymorphic STR markers were published for C. sativa [5], [6], [7] and research has shown the utility of these markers in individualizing marijuana samples [8]. However, the technique has been rarely used in crime labs due to lack of standardization and validation. An analytical method should be easy to use, standardized, and validated before it can be fully utilized by a forensic laboratory.

In order to develop a reliable STR method for cannabis identification, the best markers currently available were chosen as a measure of continuity within the field. In choosing markers, dinucleotide repeat markers were avoided. All markers chosen have been previously described using IUPAC nomenclature [9], [10]. Based upon our previous research [11], we improved upon a STR multiplex method by discarding STR loci that performed poorly and incorporating six new tetranucleotide markers recently described by Valverde et al. [9].

This paper describes the development and optimization of a C. sativa STR multiplex in addition to a comprehensive developmental validation following guidelines established by the Scientific Working Group on DNA Analysis Methods (SWGDAM) [12]. Developmental validation studies included: sensitivity, species specificity, precision and accuracy, and genetic variation in a reference population. Additionally, internal validation studies were performed to provide detailed assessments of stutter ratios, peak height ratios (PHRs), and inter-locus balance of the assay.

Section snippets

DNA samples

DNA from marijuana samples (N = 101) was extracted from three seizures at the U.S. Customs and Border Protection LSSD Southwest Regional Science Center. All samples from the seizures belong to the same population [11]. A minimum of 10 specimens were randomly sampled from each case set. For collection, individual marijuana plant fragments (stem or flowers) were isolated and DNA was extracted and quantified according to Houston et al. [11]. Briefly, plant fragments (10 mg) were homogenized using

Optimization of PCR reaction and cycling conditions

The cannabis multiplex STR system was optimized using the Type-it™ Microsatellite PCR Kit (Qiagen). Primer concentrations were titrated to ensure inner-locus balance across the 13 STR markers (Table 1). An example of an electropherogram of the novel 13 loci STR multiplex system is displayed in Fig. 1. Annealing temperatures were determined for each marker (primer set) to avoid the occurrence of null alleles. Annealing temperatures ranged from 55 °C to 66 °C (Table 1). The optimal annealing

Conclusions

The goal of this study was to develop a 13 loci cannabis STR multiplex system for forensic DNA profiling that could approach the robustness of standard STR systems used for human identification (HID). This study was able to demonstrate that this new multiplex can produce high-quality STR profiles that are comparable with standard STR HID systems. These technologies may assist the forensic community as the demand for cannabis studies either for genetic identification or intelligence purposes

Acknowledgements

This study was supported by Award No. 2015-R2-CX-0030, awarded by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice. The opinions, findings, and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect those of the Department of Justice.

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