Advanced Detection Methods of Genomic Barcodes for Genotyping Escherichia coli Libraries

Sammanfattning: Pooled cell strain libraries are a powerful tool allowing to investigate the influence of genetic modifications on phenotypes in high throughput single-cell assays. To link the genotype to phenotype in each cell of the library, unique 20 base pairs (bp) long barcodes are used to allow in situ genotyping after phenotyping via fluorescence microscopy. In previous studies, these barcode sequences were expressed from high copy number plasmids resulting in a high number of targets for detection via fluorescence in situ hybridization (FISH) and thus, a strong readout signal. However, constant selection pressure must be applied on the cells to maintain the foreign plasmid DNA which may influence the phenotype. Inserting unique barcodes on the chromosome ensures stability of the construct which is required for some genomic library applications. However, the low copy number of the barcode sequence often requires an additional step of DNA amplification for efficient detection. In this study, two methods for barcode amplification were investigated. First, amplification from the double stranded DNA upon binding of peptide nucleic acids and subsequent amplification via rolling circle amplification (AmPPR). Second, amplification from genomic DNA or cDNA via loop-mediated isothermal amplification (LAMP). Whereas the AmPPR approach remained unsuccessful, chromosomal barcode sequences were successfully amplified in situ via LAMP and subsequently detected using FISH. I show that LAMP can potentially be a quick, specific, and elegant amplification technique for in situ genotyping in microfluidic devices. However, nonspecific amplification and partly nonspecific readout signals when using LAMP remain a problem and need to be further investigated before implementing this method on pooled libraries.