Microfluidic extraction and stretching of chromosomal DNA from single cell nuclei for DNA fluorescence in situ hybridization

Abstract

We have developed a novel method for genetic characterization of single cells by integrating microfluidic stretching of chromosomal DNA and fiber fluorescence in situ hybridization (FISH). In this method, individually isolated cell nuclei were immobilized in a microchannel. Chromosomal DNA was released from the nuclei and stretched by a pressure-driven flow. We analyzed and optimized flow conditions to generate a millimeter-long band of stretched DNA from each nucleus. Telomere fiber FISH was successfully performed on the stretched chromosomal DNA. Individual telomere fiber FISH signals from single cells could be resolved and their lengths measured, demonstrating the ability of the method to quantify genetic features at the level of single cells.

Microfluidic Device for On-Chip Immunophenotyping and Cytogenetic Analysis of Rare Biological Cells

“…Liu et al [19] performed FISH on centromere-sized cell arrays modified with 3-aminopropyltriethoxysilane (APTES) or polyethylene glycol (PEG)-coated glass slides with overnight hybridization. Wang et al [20] introduced an APTES-coated glass slide with a PDMS microfluidic for stretching chromosomal DNA from a single cell to perform FISH. In a recent study, a microfluidic consisting of a Pyrex-Si stack was generated and used for FISH to provide breast cancer prognosis [21][22][23][24].…”

Ordered Arrays of Native Chromatin Molecules for High-Resolution Imaging and Analysis

“…This technique could be extended to the use of microfluidics for dealing with material extracted from a single cell. 19 …”

“…The concept of stretching nucleic acids for analysis has been largely exploited in the case of bare DNA. Current techniques for DNA stretching comprise end-tethering, in combination with optical or magnetic tweezers, 6–7 stretching on polydimethylsiloxane (PDMS) stamps, 8–13 adsorption onto a modified surface under flow, 14–15 shear flow, 16–19 and nanoconfinement. 20–21 While these techniques should be transposable to chromatin, we note that chromatin stretching is less studied, except for end-tethered stretching 22–27 and a few recent nanoconfinement studies.…”

Chromatin hierarchical branching visualized at the nanoscale by electron microscopy

“…The concept of aligning chromatin for high resolution imaging originated from naked DNA immobilization and stretching studies, which comprise of endtethering, in combination with optical or magnetic tweezers, stretching on polydimethylsiloxane (PDMS) stamps, adsorption onto a modified surface under flow, shear flow, and nanoconfinement. [28][29][30][31][32][33][34][35][36][37] Few methods have been demonstrated that allow the immobilization of isolated native chromatin molecules without disrupting their complex structure when imaged by TEM or AFM. 36 In order to facilitate high resolution imaging by EM, we developed a simple and robust method to immobilize and align isolated native chromatin on both continuous carbon coated TEM grids for positive staining EM and on holey grids for CryoEM, as shown in Figure 1, allowing finer details of chromatin to be visualized at nanoscale resolution.…”