A Simple DNA Extraction Method for Marijuana Samples Used in Amplified Fragment Length Polymorphism (AFLP) Analysis

Abstract

As a first step in developing a molecular method for the individualization of marijuana samples, we evaluated a plant DNA extraction kit. The QIAGEN plant DNeasy method uses a spin column format for recovery of DNA and is effective for obtaining high molecular weight DNA from leaf, flower (bud), and seed samples of marijuana. The average DNA yield was 125-500 ng per 100 milligrams of fresh plant tissue. The recovered DNA was of polymerase chain reaction (PCR) quality as measured by the ability to generate reproducible amplified fragment length polymorphism (AFLP) profiles. AFLP is a technique used to create a DNA profile for plant varieties and is being applied to marijuana samples by the authors to link growers and distributors of clonal material. The QIAGEN plant DNeasy method was simple, efficient, and reproducible for processing small quantities of marijuana into DNA.

Development of microsatellite markers in Cannabis sativa for DNA typing and genetic relatedness analyses

“…DNA isolation and preparation of genomic DNA Genomic DNA samples were provided by Dr Heather Coyle (Connecticut State Forensic Science Laboratory, CSFSL, USA) and Dr Gary Shutler (Royal Canadian Mounted Police, RCMP, Canada) and extracted with a QIAGEN plant DNeasy kit according to the manufacturer's recommendations [22]. Genomic DNA was digested with Sau3AI (Life Technologies, Gaithersburg, MD), a restriction endonuclease recognizing the 5'-GATC-3' DNA sequence.…”

“…Many growers of Cannabis choose selective breeding to adapt to particular growing conditions and to increase the THC content, which contributes to the intoxicating effect of Cannabis [22]. Selective breeding of high THC content plants can be obtained using two methods.…”

“…Molecular marker systems based on RAPD [20] and AFLP [21,22] have been developed and used for DNA typing analysis of C. sativa. However, RAPDs are dominant makers with poor reproducibility between labs [14].…”

Potential of marker-assisted selection in hemp genetic improvement

“…However, forensic researchers also contributed insights in the plant genetic structure that are useful for hemp breeders too. Protocols were established for the extraction of DNA for AFLP analysis from marihuana samples (Coyle et al, 2003), and RAPD (Gillan et al, 1995;Jagadish et al, 1996) and ISSR (Kojoma et al, 2002) markers were also used in an attempt to establish a more causative relation between the cannabinoid type of the plant and the markers identified. These attempts however were not fully successful, and no correlations of molecular markers with the gas chromatographic or HPLC cannabinoid's profiles of different Cannabis germplasm were detected.…”

Development of microsatellite markers in Cannabis Sativa for fingerprinting and genetic relatedness analyses

“…Many growers of Cannabis choose selective breeding to adapt to particular growing conditions and to increase the THC content, which contributes to the intoxicating effect of Cannabis [74]. Selective breeding of high THC content plants can be obtained using two methods.…”