Philippine Science Letters
vol. 3 | no. 2 | 2010
published online November 25, 2010


ARTICLE


Selection and design of new primers for multiplex polymerase chain reaction (PCR) detection of transgenes in genetically modified (GM) soybean (Glycine max L.) Roundup Ready Soybean GTS 40-3-2


by Cynthia T. Hedreyda* and Jennifer L. Roxas

National Institute of Molecular Biology and Biotechnology, College of Science, University of the Philippines, Diliman, Quezon City, Philippines 1101


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The approval for planting and direct use of several genetically modified crops in the Philippines is expected to result in requests for the certification that seeds are GM seeds for their added value or request for proof that seeds are non-GM for those who are opposed to GM food. The study was conducted to develop alternative PCR-based procedures that produce clearly distinct size amplicons that could be used to distinguish transgenes in herbicide tolerant GM Roundup Ready Soybean GTS 40-3-2. Initial multiplex PCR experiments using primers designed and reported in earlier studies could detect a 475-bp soybean lectin gene fragment and the transgenes CamV 35S promoter /EPSPS (315-bp) and nos terminator (151-bp) from GM soybean (Glycine max L.) Roundup Ready GTS 40-3-2. Detection limits for the 315-bp CamV35S/EPSPS and the 151-bp nos terminator fragments by the multiplex PCR procedure were 0.5% and 0.3%, respectively. New sets of detection primers were designed in this study based on sequences of amplicons from the initial multiplex PCR experiment in order to obtain additional primers that could be used for transgene detection. The alternative multiplex PCR using the new sets of primers and GM soybean Roundup Ready GTS 40-3-2 DNA template, resulted in different sized amplicons for the native soybean lectin gene fragment (430-bp), the CamV promoter/CP4-EPSPS (a modified form of the plant enzyme 5-enolpyruvylshikimate-3-phosphate synthase, 300-bp), and the nos terminator gene (173-bp). The new detection procedure amplified both transgenes in Roundup Ready GTS 40-3-2 soybean seed samples with distinct and clear bands when using at least 1.0% wt/wt GM soybean Roundup Ready GTS 40-3-2.


*Corresponding author
Email Address: chedreyda@mbb.upd.edu.ph
Submitted: September 5, 2010
Revised: October 28, 2010
Accepted: October 29, 2010
Published: November 25, 2010
Editor-in-charge: Eduardo A. Padlan
Reviewers:
Sevilla D. Detera-Wadleigh
Virginia D. Monje