An integrated system for enzymatic cleavage and electrostretching of freely-suspended single DNA molecules

Abstract

A novel polyacrylamide gel-based femtolitre microchamber system for performing single-molecule restriction enzyme assay on freely-suspended DNA molecules and subsequent DNA electrostretching by applying an alternating electric field has been developed. We attempted the integration by firstly initiating restriction enzyme reaction on a fluorescent-stained lambdaDNA molecule, encapsulated in a microchamber, using magnesium as an external trigger. Upon complete digestion, the cleaved DNA fragments were electrostretched to analyze the DNA lengths optically. The critical parameters for electrostretching of encapsulated DNA were investigated and optimum stretching was achieved by using 1.5 kHz pulses with electric field strength in the order of 10(3) V cm(-1) in 7% linear polyacrylamide (LPA) solution. LPA was adopted to minimize the adverse effects of ionic thermal agitation on molecular dielectrophoretic elongation in the microchamber. In our experiments, as the fragments were not immobilized throughout the entire protocol, it was found from repeated tests that digestion always occurred, producing the expected number of cleaved fragments. This versatile microchamber approach realized direct observation of these biological reactions on real-time basis at a single-molecule level. Furthermore, with the employment of porous polyacrylamide gel, the effective manipulation of DNA assays and the ability to combine conventionally independent bioanalytical processes have been demonstrated.

Beyond Gel Electrophoresis: Microfluidic Separations, Fluorescence Burst Analysis, and DNA Stretching

“…682,683 There also exists a device for sizing single DNA molecules following restriction. 711 Here, the DNA are confined to femtoliter chambers with a single DNA molecule per chamber. After the restriction digest, the DNA fragments are extended using an ac electric field applied by titanium microelectrodes.…”

Recent advances in single‐molecule detection on micro‐ and nano‐fluidic devices

“…Nakayama et al successfully combined the functions of cell lysis, protein extraction, purification, and activity assay to a single-molecule mTAS [68]. Lam et al developed an integrated system for the restriction enzymatic cleavage and subsequent electrostretching study of freely suspended single DNA molecules [69]. In a recent study, the extraction, manipulation and stretching of DNA from single human chromosomes was demonstrated on individual intergrated microfluidic devices [70].…”

Bioanalytical separations using electric field gradient techniques

“…The stretched position enables the DNA to be mechanically ligated at a known point. Another electrostretching method is used in combination with restriction enzyme assays to determine DNA fragment length [122]. After creating DNA fragments, AC DEP was used to stretch fragments ranging in length from 3 to 60 kbp in the microdevice.…”