Trevisan, Emanuele (2011) L'uso della genotipizzazione molecolare in ambito vegetale. [Laurea triennale]
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The Polymerase Chain Reaction (PCR) is a molecular biology technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Developed in 1983 by Kary Mullis, PCR is now a routinely and often indispensable technique used in medical and biological research labs for a variety of applications. These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (fingerprinting and genotyping), and the detection and diagnosis of infectious diseases. Its use in so many applications is mainly due to its flexibility, velocity and reproducibility. We developed and used three different PCR-based approaches to genotype, identify and characterize three different typology of maize mutation in spontaneous, insertional and transgenic mutants. In the first case PCR have been used to verify if a mutation in the auxin efflux carries ZmPIN1a could be responsible of the semaphore1 mutant phenotype. This spontaneous mutant present several auxin related defects and maps closely to the ZmPIN1a position. We amplify, cloned and sequenced the candidate gene coding sequence from mutant plants: based on the preliminary results no mutation has been detected in the ZmPIN1a CDS, contradicting our initial hypothesis. More successful have been the two following cases: PCR have been indeed use for the identification of epigenetic mutant obtained by Mu transposition in a segregant population and to verify the presence of transgenic plants carrying the stem corn borer resistance in a maize field. Insertional mutant genotyping has been carried out using primer combinations on the Mu terminal inverted repeats (TIR) and on the flanking gene sequences, while for GMOs detection primers have been designed on the construct cassette. These results are just the first steps into different research projects that want to study auxin transport and epigenetic regulation of maize development, but allowed us to assess the power and the plasticity of the PCR in plant research. Furthermore, the forensic genetics case clearly shows how molecular biology techniques can be useful also in not research-related circumstances.
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