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Construction of artificial micro RNA with mismatch PCR to direct silencing of lipase gene

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documents in English
case study
7 pages
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  1. Introduction
  2. Materials and methods
  3. PCR-directed mutagenesis of template clone pNW55
  4. Verification of amplified products by agarose gel electrophoresis
  5. Discussion
  6. Conclusion

Lipase is an enzyme that hydrolyzes rice bran oil into free fatty acids, causing spoilage and restricting its usage as food ingredient. Micro RNA (miRNA) is a post-transcriptional regulator endogenously produced in plants that can be potentially altered to specifically target lipase gene, silencing it in the process. This experiment aimed to design a precursor miRNA gene with mismatch primers through fusion PCR by using the rice's own miRNA osaMIR528 as template. Three separate PCR tubes, each containing specific set of primers (primer G-4368 and primer II for reaction 1; primer I and primer IV for reaction 2; primer III and primer G-4369 for reaction 3) were prepared and sent for PCR thermo-cycling. Amplified products from three reactions were combined and subjected to fusion PCR using primer G-4368 and G-4369. Amplified products from all three reactions and fusion PCR were gel electrophoresed and UV imaged. One band was observed for reactions 1, 2, and 3 (250bp, 80bp, and 250bp respectively) and was consistent with expected data.

Two bands were observed for fusion PCR with 300bp fragment being major product, disagreeing with expected result. Deletion was suspected during PCR. As conclusion, the construction of a 554bp ami RNA precursor gene for targeted knockout of rice lipase was not successful. First three PCR reactions using designed primers managed to produce desired fragment but deletion was suspected to occur in final PCR product. Further analysis had to be done to identify possible errors that caused discrepancies with expected result.

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