Restriction mapping in nanofluidic devices

Abstract

We have performed restriction mapping of DNA molecules using restriction endonucleases in nanochannels with diameters of 100 -200 nm. The location of the restriction reaction within the device is controlled by electrophoresis and diffusion of Mg 2؉ and EDTA. We have successfully used the restriction enzymes SmaI, SacI, and PacI, and have been able to measure the positions of restriction sites with a precision of Ϸ1.5 kbp in 1 min using single DNA molecules.R estriction mapping with endonucleases is a central method in molecular biology (1, 2). It is based on the measurement of fragment lengths after digestion, while possibly maintaining the respective order. We present here an approach to restriction mapping that is based on stretching DNA in nanofluidic channels, in which DNA is linearized to a length-independent fraction of its contour length (3, 4).To date, the most powerful method for constructing restriction maps of long DNA molecules (100 kbp and above) is the one developed by Schwartz and coworkers (5, 6). Their technique consists of stretching the DNA on a surface to establish a one-to-one mapping between spatial and genomic position, initiating the restriction by exposing the DNA to restriction enzymes and Mg 2ϩ , and optically observing the lengths of the resulting fragments. An elegant study of the same basic approach showing separation of specific binding and induced cutting was published by Taylor et al. (7).A fundamental principle in determining the error of a measurement, and a common strategy to reduce the experimental error, is to take multiple, statistically independent measurements of the same quantity. Note that the fixing of the molecule to the surface in Schwartz's approach prevents any fluctuations, and hence multiple molecules have to be observed to obtain statistically independent measurements of the same cut position. Moreover, genomic-length DNA molecules stretched on surfaces often exhibit inhomogeneous stretching, including breaks, and thus averaging over multiple molecules becomes imperative (6,8). In contrast, a molecule stretched inside a nanochannel is not subject to any forces other than those causing lateral confinement and thus is able to thermally relax and fluctuate around an equilibrium conformation. The evaluation of a single molecule is thus sufficient if complete digestion is achieved. A detailed description of the statistics and dynamics of DNA molecules in nanochannels is published in ref. 9.The main challenge in employing the concept of stretching and mapping DNA inside a closed fluidic system is to separate the steps of stretching and cutting. We have solved this problem by introducing the DNA electrophoretically into nanochannels and controlling the concentration of the enzyme cofactor Mg 2ϩ in the device shown in Fig. 1. It consists of a microfluidic ''loading'' channel containing the DNA to be analyzed, the restriction enzyme, and EDTA, and a microfluidic ''exit'' channel containing Mg 2ϩ and the restriction enzyme. The two microfluidic channels are linked by 10 n...

Nanofluidic structures for single biomolecule fluorescent detection

“…[42][43][44][45][46][47][48] Finally, sequence information may even be obtained from stretched molecules through restriction site mapping or motif mapping using hybridized probes. 19,20,49 In addition to stretching via hydrodynamic forces, channels, planar slits, and pillar arrays, fabricated into glass or silicon wafers can also be used to study the mechanical dynamics of a DNA molecule by employing entropic forces that arise due to confinement. To describe the origin of the confinement-induced forces that enable single molecule control and visualization, it is useful to compare the size of a DNA strand with the dimensions of nanofabricated structures.…”

“…16 As a consequence of real time length measurements, DNA molecules can be visualized as they undergo digestion by restriction enzymes (Figure 11). 19 After an enzymatic cofactor enters the nanochannel, fragments are cut by the already present enzymes and drift apart allowing for immediate length measurements. Furthermore, because fragment order is maintained, this method provides direct knowledge of cut position.…”

“…The second half of this review discusses ways in which nanofluidic structures can be used to measure the mechanical properties of single DNA molecules [15][16][17][18] and perform sequence analysis. [19][20][21][22] As with the structures that are used to confine light, the devices that perform these tasks can be placed into one of two categories. They are either ''nanoslits,'' which allow a DNA strand to exist in a quasi two-dimensional (2D) plane, or they are called ''nanochannels'' and confine the polymer to a line.…”

Enhancing Single-Nanoparticle Surface-Chemistry by Plasmonic Overheating in an Optical Trap

“…[1][2][3] Lab-on-a-chip applications frequently employ microor nanofluidic structures. [4][5][6][7] However, these approaches do not change the outcome of a chemical reaction. Optical trapping on the other hand, being compatible with micro-or nanofluidic systems, is a well-established technique that has been widely applied to singlemolecule force and optical spectroscopy, non-invasive manipulation, and chemical reactions monitoring .…”

Thermophoretic Forces on DNA Measured with a Single-Molecule Spring Balance

“…This extension exceeds all previously reported results obtained in nanochannels with similar dimensions and salt concentrations [39,40] and, hence, may be of practical interest: Research on genomic-length DNA confined inside nanofluidic structures has increased our understanding of biological interactions of DNA and enables coarse-grained optical mapping of the sequence of intact individual DNA molecules [41][42][43][44][45][46]. Stretching DNA to nearly its contour length remains a challenge, however.…”