Multiplex single-molecule DNA barcoding using an oligonucleotide ligation assay

Detection of specific nucleic acid sequences is invaluable in biological studies such as genetic disease diagnostics and genome profiling. Here we developed a highly sensitive and specific detection method that combines an advanced oligonucleotide ligation assay (OLA) with multi-color single-molecule fluorescence. We demonstrated that under our experimental conditions 7-nt long DNA barcodes have the optimal short length to ascertain specificity while being long enough for sufficient ligation. Using four spectrally separated fluorophores to label DNA barcodes, we simultaneously distinguished four DNA target sequences differing by only a single nucleotide.

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