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الكشف عن الرز المحور وراثيا باستخدام انواع مختلفة من التفاعل الانزيمي المتسلسل PCR == Detection of Genetically Modified Rice By Different Type of PCR
Author name:
ياسمين ابراهيم فرحان
Supervisor name:
امنة نعمة الثويني
General topic:
Biology
Specific topic:
Biotechnologies
Degree:
Master
University:
University of Baghdad - Institute Of Genetic Engineering And Biotechnology - Department Of Biotechnology
Language:
English
University location:
Baghdad
First pages:
24T2669 - p.pdf
Abstract:
In recent years, foods produced by genetic engineering technology have been on the world food markets. The biosafety aspects, regulations, and labeling for these foods are still contentious issues in most countries. Thus detection and quantificationof GMOs play crucial role for developing regulations on GM foods.In this study, eighty six non - labeled rice samples from different locals and exported market were analyzed to detect the genetic modification using a DNA based detectionvmethods as, conventional Polymerase chain reaction (PCR), and Real time PCR (RTPCR).The DNA rice samples were extracted by manual C - hexadecyl - Trimethyl - Ammonium - Bromide (CTAB) method and wizard kit method. The result revealed that DNA yield by the two methods is comparable. Rice DNA tends to be of a higher concentration when purified with the CTAB method; however, this particular DNA is more easily to amplify, the optical density (OD) was recorded 1.70 - 1.98 and the concentration of DNA quantified by fluorometer DNA rice samples, ranged from 11 to 50.5 ?g/?l. The DNA rice sample has also been used successfully with the Wizard Genomic DNA Purification Kit, and showed varieties in quality, the OD was recorded 1.65 - 1.95, and the concentration between 4.7 - 43.8 ?g/?l.The rice specific gene (sps gene) was detected by PCR. The results demonstrate that the purity of the extracted DNA in all tested rice samples was sufficiently high for a sensitive PCR analysis and the primer of detected gene appeared clearly at 251pb.Three genes; CaMV 35S promoter, NOS terminator, and insecticide resistant gene Cry1Ac were used to detect of GM rice by PCR, and Real time PCR using oligonucleotide sets targeting to novel genes. The result showed that there was no positive result reaction with conventional PCR, while the outcome of gradient PCR revealed a positive reaction in one sample (Uncle Bens brown) for CaMV35S promoter only. Gradient PCR with 12 replicons for each sample was used for qualitative detection of CaMV35S promoter gene, after optimization of melting temperature and cycles run (45 cycles) , the results appeared positive in the last three grades (63.9, 64.6, 64.9) for CaMV35S promoter, but NOS terminator, and CryIAc were recorded negative results.The result of Real - Time PCR clarified that the CaMV35S promoter specific primer showed strong amplification with Ct, and Tm values were reached into 33.73, 38.63 and 61.55, 62.92 in two samples Uncle Bens brown and Himalayan brown, respectively, whereas NOS terminator gave positive results in four samples Maxims, Laasturiana, Carolin white and Mahatma, and the values Ct and Tm reached to30.87, 30.31, 30.54, 33.75 and 64.53, 64.61, 62.62, 63.87 respectively in comparison with the positive control, while CryI Ac which did not show any positive signal.It was concluded that using molecular methods like Real - time PCR will be useful tool for detecting GM rice such as a part of the approval detection processes because of the rarity of data concerning consumption of GM rice in Iraq.
