Multiplex Single-Molecule DNA Barcoding Using an Oligonucleotide Ligation Assay

Ivo Severins, Malwina Szczepaniak, Chirlmin Joo

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

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.

Original languageEnglish
Pages (from-to)957-967
Number of pages11
JournalBiophysical Journal
Volume115
Issue number6
DOIs
StatePublished - 18 Sep 2018

Bibliographical note

Funding Information:
This work was financially supported by grants from the European Research Council under the European Union’s Seventh Framework Programme ( FP7/2007-2013 ) and European Research Council grant agreement no. 309509 . A preliminary version of this work, https://doi.org/10.1101/265215 , was deposited in bioRxiv on February 13, 2018.

Funding Information:
This work was financially supported by grants from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013) and European Research Council grant agreement no. 309509. A preliminary version of this work, https://doi.org/10.1101/265215, was deposited in bioRxiv on February 13, 2018.

Publisher Copyright:
© 2018 Biophysical Society

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