Manuel Porcar1, Silvia Ramos, Amparo Latorre. 1. Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de València, Apartado Postal 22085, 46071 València, Spain.
Abstract
BACKGROUND: The polymerase chain reaction (PCR) is a powerful tool that is being increasingly used for detection of transgenic DNA. PCR requires only a minute quantity of template, but sensitive and accurate testing requires DNA of sufficient purity and free from inhibitors such as plant polysaccharides. Several standard protocols are available for this purpose, but they usually involve several steps, imply destruction of the maize kernel, or are time-consuming. Our aim was to develop a fast and simple extraction method to isolate a raw DNA-containing solution from maize tissues suitable for use as a template in a PCR-based detection assay with specific oligonucleotides directed to the identification of event MON810. RESULTS: The NaOH-based DNA extraction method we report here is time-saving (5 min) and can be used to isolate DNA-containing solutions from a small maize leaf portion (down to 1 mg) or from a single overnight-germinated kernel. PCR performed with selected primers yielded reproducible detection of transgenic DNA. CONCLUSION: The main advantages of the procedure are the quick extraction step, the possibility of non-destructive testing of maize kernels, and the robustness of the PCR-based detection, a consequence of the selection of MON810-matching oligonucleotides yielding intense and highly specific amplicons.
BACKGROUND: The polymerase chain reaction (PCR) is a powerful tool that is being increasingly used for detection of transgenic DNA. PCR requires only a minute quantity of template, but sensitive and accurate testing requires DNA of sufficient purity and free from inhibitors such as plant polysaccharides. Several standard protocols are available for this purpose, but they usually involve several steps, imply destruction of the maize kernel, or are time-consuming. Our aim was to develop a fast and simple extraction method to isolate a raw DNA-containing solution from maize tissues suitable for use as a template in a PCR-based detection assay with specific oligonucleotides directed to the identification of event MON810. RESULTS: The NaOH-based DNA extraction method we report here is time-saving (5 min) and can be used to isolate DNA-containing solutions from a small maize leaf portion (down to 1 mg) or from a single overnight-germinated kernel. PCR performed with selected primers yielded reproducible detection of transgenic DNA. CONCLUSION: The main advantages of the procedure are the quick extraction step, the possibility of non-destructive testing of maize kernels, and the robustness of the PCR-based detection, a consequence of the selection of MON810-matching oligonucleotides yielding intense and highly specific amplicons.