Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes.

Brian J Beliveau,Ruth B Mccole,Peng Yin,Chao-Ting Wu,Chamith Y Fonseka,Jelena Erceg,Caroline Kim-Kiselak,Jeannie T Lee,William M Shih,Xiaowei Zhuang,Frédéric Bantignies,Ralf Jungmann,Hien G Hoang,Alistair N Boettiger,Mohammed A Hannan,David Colognori,Eric F Joyce,Maier S Avendaño

doi:10.1038/ncomms8147

Abstract

Fluorescence in situ hybridization (FISH) is a powerful single-cell technique for studying nuclear structure and organization. Here we report two advances in FISH-based imaging. We first describe the in situ visualization of single-copy regions of the genome using two single-molecule super-resolution methodologies. We then introduce a robust and reliable system that harnesses single-nucleotide polymorphisms (SNPs) to visually distinguish the maternal and paternal homologous chromosomes in mammalian and insect systems. Both of these new technologies are enabled by renewable, bioinformatically designed, oligonucleotide-based Oligopaint probes, which we augment with a strategy that uses secondary oligonucleotides (oligos) to produce and enhance fluorescent signals. These advances should substantially expand the capability to query parent-of-origin-specific chromosome positioning and gene expression on a cell-by-cell basis.

Highlights

We report two strategies for in situ single-cell imaging, one that facilitates two forms of single-molecule super-resolution microscopy and another that utilizes single-nucleotide polymorphisms (SNPs) to visually distinguish homologous chromosomal regions. Both make use of Oligopaints, which are highly efficient, renewable, strand-specific fluorescence in situ hybridization (FISH) probes derived from complex singlestranded DNA libraries in which each oligo carries a short stretch of homology to the genome (Fig. 1a)
Central to the design of Oligopaints is the inclusion of non-genomic sequences flanking the region of homology to the genome, as these sequences enable the amplification by PCR or other methods to produce DNA or RNA oligos, introduction of label and conversion of doublestranded to single-stranded products[7] (Fig. 1a)
We began our current studies by examining the ability of MainStreet to recruit a fluorescently labelled ‘secondary’ oligo, as we were intrigued by the potential of secondary oligos to simplify the use of multiplexed Oligopaint libraries

Summary

Introduction

We introduce a robust and reliable system that harnesses single-nucleotide polymorphisms (SNPs) to visually distinguish the maternal and paternal homologous chromosomes in mammalian and insect systems Both of these new technologies are enabled by renewable, bioinformatically designed, oligonucleotide-based Oligopaint probes, which we augment with a strategy that uses secondary oligonucleotides (oligos) to produce and enhance fluorescent signals. We report two strategies for in situ single-cell imaging, one that facilitates two forms of single-molecule super-resolution microscopy and another that utilizes SNPs to visually distinguish homologous chromosomal regions Both make use of Oligopaints, which are highly efficient, renewable, strand-specific fluorescence in situ hybridization (FISH) probes derived from complex singlestranded DNA (ssDNA) libraries in which each oligo carries a short stretch of homology to the genome (Fig. 1a). Our studies take advantage of two features of Oligopaints: the inclusion of non-genomic sequences, which enable super-resolution imaging, and a programmable insert of genomic homology, which makes it possible for Oligopaints to bind at SNPs

Methods

Results

Conclusion