Lynch syndrome is an autosomal dominant inherited disorder characterized by an increased risk of developing various types of cancer. The condition is caused by
mutations in genes involved in the DNA mismatch repair (MMR) system, with MLH1 being one of the most frequently affected genes. Mutations in MLH1 often lead to
impaired interaction with its protein partner PMS2, which is why accurate identification of pathogenic variants of MLH1 is essential for diagnosis. One of the key in vivo
methods for studying protein-protein interactions is theyeast two-hybrid (Y2H) system, which is also the central method used in this master's thesis. Y2H is base on the fusion of the protein of interest to separate domains of a transcription factor. If the proteins interact, the transcription factor is reconstituted and activates a reporter system. The aim of this work was to construct an allele library of MLH1 that may potentially contribute
to the development of Lynch syndrome. If interaction between MLH1 and PMS2 is observed using Y2H, the mutation is likely non-pathogenic. To this end, a library of
random MLH1 variants was generated using six degenerate codons introduced by PCR method across five synthetic fragments. The integrity of the library assembly was
verified by Sanger sequencing, where we found that in vitro PCR-based assembly was more effective than in vivo recombination of fragments. Following the optimization of
transformation protocol, we scaled up the Y2H assay in liquid media, isolated plasmids,
amplified the variable region and submitted samples for amplicon sequencing. A subsequent bioinformatic analysis confirmed the effectiveness of the selection strategy,
but also revealed that the actual diversity of the library was much lower than its theoretical potential. We found that most pathogenic mutations were located at codons 636 and 661 that are situated deeper within the gene structure.
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