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...
Recently developed DNA-modified diamond surfaces exhibit excellent chemical stability to high-temperature incubations in biological buffers. The stability of these surfaces is substantially greater than that of gold or silicon surfaces, using similar surface attachment chemistry. The DNA molecules attached to the diamond surfaces are accessible to enzymes and can be modified in surface enzymatic reactions. An important application of these surfaces is for surface invasive cleavage reactions, in which target DNA strands added to the solution may result in specific cleavage of surface-bound probe oligonucleotides, permitting analysis of single nucleotide polymorphisms (SNPs). Our previous work demonstrated the feasibility of performing such cleavage reactions on planar gold surfaces using PCR-amplified human genomic DNA as target. The sensitivity of detection in this earlier work was substantially limited by a lack of stability of the gold surface employed. In the present work, detection sensitivity is improved by a factor of approximately 100 (100 amole of DNA target compared with 10 fmole in the earlier work) by replacing the DNA-modified gold surface with a more stable DNA-modified diamond surface.
The structure-specific invasive cleavage reaction is a useful means for sensitive and specific detection of single nucleotide polymorphisms, or SNPs, directly from genomic DNA without a need for prior target amplification. A new approach integrating this invasive cleavage assay and surface DNA array technology has been developed for potentially large-scale SNP scoring in a parallel format. Two surface invasive cleavage reaction strategies were designed and implemented for a model SNP system in codon 158 of the human ApoE gene. The upstream oligonucleotide, which is required for the invasive cleavage reaction, is either co-immobilized on the surface along with the probe oligonucleotide or alternatively added in solution. The ability of this approach to unambiguously discriminate a single base difference was demonstrated using PCR-amplified human genomic DNA. A theoretical model relating the surface fluorescence intensity to the progress of the invasive cleavage reaction was developed and agreed well with experimental results.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.