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Submitted to: Cereal Chemistry
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 7/16/2003 Publication Date: 1/12/2004 Citation: TILLEY, M. 2004. PCR AMPLIFICATION OF WHEAT SEQUENCES FROM DNA EXTRACTED DURING MILLING AND BAKING. CEREAL CHEMISTRY. 81(1):44-47. Interpretive Summary: The labeling of genetically modified foods is dependent on the development of methods that are capable of sensitive and accurate detection. Detection of transgenic events can be based on the detection of novel proteins produced by the genetically modified organism or the detection of the modified DNA itself. DNA-based analysis is the method of choice due to the fact that DNA is very stable and very sensitive and accurate analytical techniques are available. We have used DNA analysis to determine the effects of various steps in the bread baking process on wheat DNA. DNA samples were extracted from Hard Red Winter wheat kernels, milling fractions, flour, fully mixed dough, 1st punch, 2nd punch, moulding, pan stages, during the bake (at 5, 10, and 15 minutes), and after the bake (at 1, 3, and 5 days). DNA purified from whole kernels demonstrated the expected long, high molecular weight strands containing over 12,000 base pairs. DNA extracted from the flour was partially broken into smaller pieces ranging in size from less than 300 base pairs to the original >12,000 base pairs. The majority of the large DNA pieces were contained in the bran fractions. During the first ten minutes of the baking process, DNA fragmentation accelerates. DNA purified from bread contained a maximum size of 400 base pairs. These results show that, as wheat is processed into flour and baked, the DNA molecules are broken into smaller and smaller pieces. However, the DNA-based analytical method used is sensitive enough to detect low levels of genetic materials in finished bakery products and should be able to detect the presence of transgenic genes. It is interesting to note that low levels of yeast DNA also were detected in the final bread products. Technical Abstract: Detection of transgenic events are based upon the identification of novel proteins specific activities, detection of specific DNA sequences. Processing steps have a profound effect upon the proteins and DNA present in the final product. DNA-based analysis has several advantages over protein-based methods due to the fact that DNA is highly stable and DNA-based analyses are highly sensitive and specific. This project examined the effects of breadmaking on wheat DNA extracted from various steps in the baking process. Samples were taken from wheat kernels, milling fractions, flour, and at steps during and after the baking process. Kernel DNA contained high molecular weight DNA (>12,000 base pairs - bp), whereas that from flour exhibited a broad smear (>12,000 bp to <300 bp). The polymerase chain reaction (PCR) was used to amplify sequences present at different copy numbers within the wheat genome. PCR successfully amplified products of both high and low copy number, however, successful amplification requires that the maximum size be no more than the average molecular weight of the DNA recovered from the source. The data also demonstrated the ability to detect the presence of a minor ingredient (yeast). |
