ORIGINAL ARTICLE DNA Analysis of Digested Tomato Seeds in Stomach Contents Cheng-Lung Lee,*† Heather Miller Coyle,‡ Eric Carita,§ Carll Ladd,§ Nicholas C.S. Yang,§ Timothy M. Palmbach,‡ Ian C. Hsu,* and Henry C. Lee, PhD‡§ Abstract: 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 morpho- logic 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 identifica- tion. 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. Key Words: forensic science, forensic botany, stomach content, tomato seed, seed identification, plant DNA, amplified fragment length polymorphism (Am J Forensic Med Pathol 2006;27: 121–125) T he collection and examination of stomach contents and the contents of the small intestine and large intestine are important steps in medical legal autopsy. The results of such examination often provide important information for forensic purposes. Identification of seeds from stomach contents can verify the decedent’s last meal, provide linkages between victim and suspect, or use traceable seed evidence in death investigations. 1,2 In addition, stomach contents collected at autopsy may contain identifiable plants and seeds that can be used for estimating the time of death 3 or verifying an alibi. 1 Several methods have been reported for the identification of plant species in gastric contents. Hayashiba et al 4 and Nagata et al 5 have reported an HPLC (high-performance liquid chro- matography) technique to identify vegetable species in gastric contents. However, the majority of medical examiners and forensic scientists still rely on the microscopic and morpho- logic methods to identify plant and food materials from stomach contents. 1,6,7 This study reports on the development of a new biologic method, DNA typing by amplified fragment length polymorphism (AFLP) to further individualize seeds after a microscopic identification of the plant species has been performed. AFLP markers were developed by Vos et al 8 and represent a method for genotyping single-source plant sam- ples for a large number of markers to generate a highly discriminating DNA band pattern. The AFLP technique is based on the detection of genomic restriction fragments by PCR amplification. Band patterns are produced without prior sequence knowledge, using a limited set of amplification primers. Figure 1 is a schematic diagram which illustrates the general procedure. This technique has been used to identify fresh tomato samples but not for ingested samples. 9 –11 This PCR-based technique permits the inspection of polymor- phisms at a large number of loci within a short period of time and requires only a small amount of DNA (about 10 ng). Although other DNA typing techniques exist and have been developed for specific applications, 12–15 AFLP analysis is the method of choice since it has general applicability to any single-source plant sample, regardless of the species. 8,16,17 Many edible fruits and vegetables contain seeds with tough, durable seed coats that remain intact after passing through the human digestive system. DNA analysis of di- gested seeds to individualize the source of the seed sample is a novel area of research. If a DNA test of a digested seed is feasible, then genetic profiles (DNA databases of variety- specific markers) can be established to identify and charac- terize different cultivars of edible plant and seed spe- cies. 8,18,19 This study was designed to determine if the recovered tomato seed DNA was useful for DNA typing by the AFLP method. Manuscript received March 23, 2005; accepted April 5, 2005. From the *Nuclear Science Department, National Tsing Hua University, Hsinchu, Taiwan, ROC; †Criminal Investigation Department, Hsinchu Municipal Police Bureau, Hsinchu, Taiwan, ROC; ‡Forensic Science Program, University of New Haven, West Haven, Connecticut; and the §Division of Scientific Services, Department of Public Safety, 278 Colony Street, Meriden, Connecticut. Sources of scholarship support: National Science Council, ROC #NSC92- 2917-1-007-004; National Institute of Justice #2001-IJ-CX-K011. No human or animal studies were performed without appropriate consent. Reprints: Henry C. Lee, PhD, Division of Scientific Services, Department of Public Safety, 278 Colony Street, Meriden, CT 06451. E-mail c/o Ms. Valerie Shook: valerie.shook@po.state.ct.us. Copyright © 2006 by Lippincott Williams & Wilkins ISSN: 0195-7910/06/2702-0121 DOI: 10.1097/01.paf.0000202722.21167.47 The American Journal of Forensic Medicine and Pathology • Volume 27, Number 2, June 2006 121