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Biophys J. 2018 Sep 18;115(6):957-967. doi: 10.1016/j.bpj.2018.08.013. Epub 2018 Aug 18.

Multiplex Single-Molecule DNA Barcoding Using an Oligonucleotide Ligation Assay.

Author information

1
Kavli Institute of NanoScience, Department of BioNanoScience, Delft University of Technology, Delft, The Netherlands.
2
Kavli Institute of NanoScience, Department of BioNanoScience, Delft University of Technology, Delft, The Netherlands. Electronic address: malwina.szczepaniak@gmail.com.
3
Kavli Institute of NanoScience, Department of BioNanoScience, Delft University of Technology, Delft, The Netherlands. Electronic address: c.joo@tudelft.nl.

Abstract

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 with multicolor single-molecule fluorescence. We demonstrated that under our experimental conditions, 7-nucleotide 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. Our single-molecule approach will allow for accurate identification of low-abundance molecules without the need for target DNA preamplification.

PMID:
30195940
PMCID:
PMC6139880
DOI:
10.1016/j.bpj.2018.08.013

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