Heather Miller Coyle

Field Testing of Collection Cards for Cannabis sativa Samples With a Single Hexanucleotide DNA Marker

Abstract:  The validity and feasibility of using DNA collection cards in the field for preservation and analysis of Cannabis sativa genotypes were investigated using a highly specific hexanucleotide marker. Collection cards were submitted to the National Marijuana Initiative, which selectively trained and managed the collection of specific types of samples from a variety of participating agencies. Samples collected at seizure sites included fresh marijuana leaf samples, dried “dispensary” samples, U.S. border seizures, and hashish. Using a standardized PCR kit with custom‐labeled oligonucleotide primers specific to marijuana, collection cards produced eight genotypes and 13 different alleles, extremely low baselines, and no cross‐reactivity with control plant species. Results were produced from all sample types with the exception of hashish. Plant DNA collection cards represent an easily implementable method for the genetic identification and relatedness of C. sativa street and grow site–seized samples with applications for databasing and market disruption.

Analysis of the NMI01 marker for a population database of cannabis seeds.

We have analyzed the distribution of genotypes at a single hexanucleotide short tandem repeat (STR) locus in a Cannabis sativa seed database along with seed‐packaging information. This STR locus is defined by the polymerase chain reaction amplification primers CS1F and CS1R and is referred to as NMI01 (for National Marijuana Initiative) in our study. The population database consists of seed seizures of two categories: seed samples from labeled and unlabeled packages regarding seed bank source. Of a population database of 93 processed seeds including 12 labeled Cannabis varieties, the observed genotypes generated from single seeds exhibited between one and three peaks (potentially six alleles if in homozygous state). The total number of observed genotypes was 54 making this marker highly specific and highly individualizing even among seeds of common lineage. Cluster analysis associated many but not all of the handwritten labeled seed varieties tested to date as well as the National Park seizure to our known reference database containing Mr. Nice Seedbank and Sensi Seeds commercially packaged reference samples.

DNA Analysis of Digested Tomato Seeds in Stomach Contents

Examination of stomach contents is one of the important steps in medical legal autopsy. Vegetative materials such as stems, roots, and seeds in stomach contents can be valuable evidence for providing investigative leads in death investigation. Currently, the identification of plant materials relies on microscopic and morphologic examination. We have found that many seeds are often protected from acid degradation during stomach digestion by their tough exterior seed coat. Tomato seeds were selected as a model system to assess DNA analysis and plant variety marker identification. The DNA-amplified fragment length polymorphism method was performed to determine if the DNA obtained from single seeds could be used for PCR analysis. From the amplified fragment length polymorphism results, some candidate markers for individualizing seeds from morphologically distinct tomatoes were identified. These data on DNA analysis of tomato seeds indicate amplified fragment length polymorphism is a viable procedure for the individualization of seeds from stomach contents in forensic investigations.

DNA analysis of ingested tomato and pepper seeds.

Ingested food is one of the important types of forensic evidence obtained during a medicolegal autopsy. Many materials containing seeds pass through the human digestive system and are still recognizable; thus, they can be valuable for providing investigative leads. Currently, the identification of seeds relies on microscopic and morphologic examination. However this method sometimes can be problematic. For example, the microscopic appearance of the ingested tomato and pepper seeds is very similar; thus, it is not always easy to distinguish these seeds by comparing their physical characteristics. Tomato and pepper seeds were selected as a model system to assess the value of performing DNA analysis as an alternate and/or complimentary means of seed identification. Results of blind testing indicate that the deoxyribonucleic acid-amplified fragment length polymorphism (DNA-AFLP) results were able to discriminate between pepper and tomato seed samples after they passed through the digestive system.

The importance of scientific evaluation of biological evidence — Data from eight years of case review

In 2009, the National Research Council published a report stating that the addition of more science and technology into the field of forensic science in the United States would be of great benefit to the judicial system. As a starting point to address this NRC report, one needs to make an assessment of the system. One factor that is continuously requested is an estimate of an error rate. In any given scientific area of forensics that is difficult to quantitate except by external review and audits. After eight years of requested defense review of cases with biological and DNA evidence, most cases appear to be scientifically sound in test methods and procedures. However, there were some cases where errors in the forensic science process did occur. This article takes information compiled from those eight years of defense review and summarizes the cases where errors have been discovered and discusses the scientific implications of these errors. The scope of this article is limited to crime scene collection and forensic science laboratory testing of biological materials for body fluid identification and DNA individualization to a source. The greatest value of defense review comes from (a) providing effective balance and independent oversight to the judicial process and (b) collecting data into a format that can be useful as a guide in training programs.

Capillary Electrophoresis of DNA from Cannabis sativa for Correlation of Samples to Geographic Origin

For routine genetic analysis of Cannabis sativa, two methods are currently in use, (a) AFLP; amplified fragment length polymorphism analysis and (b) STR; short tandem repeat analysis. The AFLP method used on capillary electrophoresis instrumentation is fully described in this chapter. AFLP analysis generates numerous nonspecific marker fragments for a complex DNA pattern and is available in kit format for quality assurance of reagents. This method is particularly useful when discerning the genetics of highly inbred plant species that may share much of the same DNA with only slight differences due to their common genetic background. AFLP analysis, however, is most effective on fresh or well-preserved plant specimens where the integrity of the DNA is high and the sample is a single source specimen (i.e., not a mixture of plants or different species).