DNA bridges: A novel platform for single-molecule sequencing and other DNA-protein interaction applications

Abstract

DNA molecular combing is a technique that stretches thousands of long individual DNA molecules (up to 10 Mbp) into a parallel configuration on surface. It has previously been proposed to sequence these molecules by synthesis. However, this approach poses two critical challenges: 1-Combed DNA molecules are overstretched and therefore a nonoptimal substrate for polymerase extension. 2-The combing surface sterically impedes full enzymatic access to the DNA backbone. Here, we introduce a novel approach that attaches thousands of molecules to a removable surface, with a tunable stretching factor. Next, we dissolve portions of the surface, leaving the DNA molecules suspended as ‘bridges’. We demonstrate that the suspended molecules are enzymatically accessible, and we have used an enzyme to incorporate labeled nucleotides, as predicted by the specific molecular sequence. Our results suggest that this novel platform is a promising candidate to achieve high-throughput sequencing of Mbp-long molecules, which could have additional genomic applications, such as the study of other protein-DNA interactions.

Microfluidic delivery of cutting enzymes for fragmentation of surface-adsorbed DNA molecules

“…Also, patterned surfaces (polyethylene glycol-octenyl silane [ 76 ] with variable adsorption behavior may be useful. Recently, Righini et al [ 77 ] have developed a lithographic technique to permit stretched DNAs to be suspended over corrugated phoresist surfaces, reducing steric hindrance and providing an open structure which can facilitate desorption. Microporous substrates [ 78 , 79 ] may also prove useful in this regard.…”

Molecular combing and its application in clinical settings

“…As this procedure does not cause modifications in DNA sequence, it provides new possibilities to study the structure of DNA and especially the order of genes and loci. In the following substrate, straightening mechanism, pH condition, tension size were studied in detail to improve MCT [ 13 ].

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“…In interphases DNA is more decondensed, still the order of three closely localized genes can only be determined reliably when evaluating 20–50 cells in a semi-statistical way; besides, the distance between them has to be in the range of 0.5 to 1 Mb or more. To achieve higher resolutions, approaches like fiber-FISH or molecular combing technique (MCT) were established [ 10 – 13 ]. In this review, MCT principle and how to perform, applications in medical field, advantages and shortcuts are presented and discussed.…”

Driving Forces for Single DNA Stretching Assessed by In Situ TIRFM

“…DNA, a critical hereditary matter in humans and almost all other organisms, stores a large quantities of genetic information. , The study of the structure-related properties of DNA molecules has been a fascinating and intriguing topic. , However, coiled DNA molecules randomly dispersed in solution make it extremely challenging to analyze their physical features and decipher their gene code, especially in a liquid environment. − It is critical to develop effective single-molecule manipulation techniques for DNA stretching to extract the DNA structural information and to further study the genetics of DNA molecules. In the last two decades, a variety of DNA manipulation techniques have been proposed and developed, such as magnetic tweezers, − optical tweezers, , hydrodynamic flow stretching, − molecular combing, − etc., giving opportunities to study the dynamic behaviors of DNA molecules and other important characteristics.…”