Lectin gene detection as control for GMO assay by quantitative competitive PCR | |
| Author | Acharya, Rupa Aryal |
| Call Number | AIT Thesis no.BT-03-17 |
| Subject(s) | Transgenic plants Lectins Quantitative genetics |
| Note | A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science, School of Environment, Resources and Development |
| Publisher | Asian Institute of Technology |
| Series Statement | Thesis ; no. BT-03-17 |
| Abstract | Detection and quantification of genetically modified organisms in the foods has been mandatory for the labeling of the foods so that the consumers can get the foods of their choice with required information. DNA extraction has been the most critical step for successful GMO detection and quantification. The PCR is the main downstream process of GMO detection, which is dependent totally on the quality, and purity of the DNA. So different extraction techniques were studied and the most effective, reliable and easier technique was used for further quantification of Roundup Ready Soybean by QC-PCR. Among the four methods tested CT AB conventional method worked well in this study providing good performance characteristic in PCR of the genes of interest. Sequencing of lectin gene was carried out to determine the exact open reading frame of the lectin gene. For QC-PCR internal standard lectin was constructed using the Recombinant PCR technique with the help of the mutagenic primers designed by insertion of artificial bases in the middle of the target gene. The constructed standard was calibrated for the GMO equivalence and was found that 500ng of target DNA of 1 % RRS soya flour and lμg of 1 % RRS tempe have the equivalent intensity with the 0.322pg and 0.296pg of internal standard lectin respectively. QC-PCR calibration was carried out using the internal standard 35S and 1 % RRS soya flour and the point of equivalence was obtained in terms of intensity when 500ng of 1 %RRS soya flour co amplified with 4.25x10-6pg of the internal standard. For the quantification of Roundup Ready soybean in the tempe samples the ratio of 35S promoter and the Soya specific lectin gene could not be taken as the index as tempe failed to amplify in the QC-PCR for 35S. |
| Year | 2003 |
| Corresponding Series Added Entry | Asian Institute of Technology. Thesis ; no. BT-03-17 |
| Type | Thesis |
| School | School of Environment, Resources, and Development (SERD) |
| Department | Department of Food, Agriculture and Natural Resources (Former title: Department of Food Agriculture, and BioResources (DFAB)) |
| Academic Program/FoS | Food Engineering and Bioprocess Technology (FB) |
| Chairperson(s) | Stevens, Willem F.; |
| Examination Committee(s) | Sunee Nitisinprasert;Suwalee Chandrkrachung;Yang Yi; |
| Scholarship Donor(s) | The Royal Netherlands Government; |
| Degree | Thesis (M.Sc.) - Asian Institute of Technology, 2003 |