Development of a competitive polymerase chain reaction technique for GMO quantification | |
| Author | Tiwari, Krishna Raj Singh |
| Call Number | AIT Thesis no. BP-02-12 |
| Subject(s) | Transgenic organisms Polymerase chain reaction |
| Note | A thesis submitted in partial fulfillment of the requirement for the degree of Master of Science, School of Environment, Resources and Development |
| Publisher | Asian Institute of Technology |
| Series Statement | Thesis no. BP-02-12 |
| Abstract | Genetically modified organisms (GMOs) has been in debate for the public acceptance. The application of the recombinant DNA technology has resulted in the development of GM Os as promising solutions for food production and fighting pest problems. But on the other hand their conjectural risk of biological hazards forced the authorities to put regulations on the GMO labeling as being an informed choice on its use for the general public. The GMO quantification using QC-PCR techniques targeting the 35S promoter enables the detection and quantification of most of the approved GM Os in the market. The QC-PCR is the co-amplification of a known amount of internal standard GMO DNA with the unknown quantity of target GMO DNA. Thus with a proper calibration it is possible to detect the absolute % of GMO content in the sample. The use of an internal standard also reduces the interlaboratory differences in their results. The internal standard for the 35S promoter was prepared by insertion of an extra oligonucleotide in the middle of the 35S primer annealing site. The middle insertion in the 35S promoter was achieved by amplifying two half of the 35S segment from two designed middle primers. The middle primers with extra insertional sequence were used with outer 35S primer to amplify the two half modified segments having complementary among them. The recombinant PCR was done to ligate them by recombination and extension with Taq. Thus constructed standard segment was cloned to pGEM-T vector and transformed to E.coli. The cloned plasmids were calibrated as internal standard for the equivalence amount of the various GMO% using standard GMO sample. The calibration was done by comparing the band intensity on the electrophoresis gel with the help of image analysis software. For the GMO quantification of the sample the different amount of the standard, equivalent to various GMO% was co-amplified using the same amount of the unknown target template. The resulting band intensity of target and standard was analysed with the help of Scion® Image software. The target giving equal band intensity with the standard corresponds to the % GMO content in the sample. |
| Year | 2002 |
| Corresponding Series Added Entry | Asian Institute of Technology. Thesis ; no. BP-02-12 |
| 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 | Bioprocess Technology (BP) |
| Chairperson(s) | Stevens, Willem F.; |
| Examination Committee(s) | Suwalee Chandrkrachang ;Sunee Nitisinprasert ;Mair, Graham C.; |
| Scholarship Donor(s) | The Royal Netherlands Government; |
| Degree | Thesis (M.Sc.) - Asian Institute of Technology, 2002 |