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Highly sensitive restriction enzyme assay and analysis: a review

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Abstract

Biological assays at the single molecule level are crucial to fundamental studies of DNA-protein mechanisms. In order to cater for high throughput applications, one area of immense research potential is single-molecule bioassays where miniaturized devices are developed to perform rapid and effective biological reactions and analyses. With the success of various emerging technologies for engineering miniaturized structures down to the nanoscale level, supported by specialized equipment for detection, many investigations in the field of life science that were once thought impossible can now be actively explored. In this review, the significance of downscaling to the single-molecule level is firstly presented in selected examples, with the focus placed on restriction enzyme assays. To determine the effectiveness of single-molecule restriction enzyme reactions, simple and direct analytical methods based on DNA stretching have often been reliably employed. DNA stretching can be realized based on a number of working principles related to the physical forces exerted on the DNA samples. We then discuss two examples of a nanochannel system and a microchamber system where single-molecule restriction enzyme digestion and DNA stretching have been integrated, which possess prospective capabilities of developing into highly sensitive and high-throughput restriction enzyme assays. Finally, we take a brief look at the general trends in technological development in this field by comparing the advantages and disadvantages of performing assays at bulk, microscale and single-molecule levels.

Minaturization of Restriction Enzyme Assays and DNA Stretching

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Abbreviations

DNA:

Deoxyribonucleic acid

PAA:

Polyacrylamide

LPA:

Linear polyacrylamide

CCD:

Charge-coupled device

λDNA:

Lambda deoxyribonucleic acid

LIF:

Laser-induced fluorescence

AFM:

Atomic force microscopy

ssDNA:

Single-stranded deoxyribonucleic acid

AC:

Alternating current

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Authors and Affiliations

  1. The Institute of Scientific and Industrial Research, Osaka University, Osaka, 567-0047, Japan

    Liza Lam, Ryota Iino, Kazuhito V. Tabata & Hiroyuki Noji

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  1. Liza Lam
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  2. Ryota Iino
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  3. Kazuhito V. Tabata
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  4. Hiroyuki Noji
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Correspondence to Liza Lam or Hiroyuki Noji.

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Lam, L., Iino, R., Tabata, K.V. et al. Highly sensitive restriction enzyme assay and analysis: a review. Anal Bioanal Chem 391, 2423–2432 (2008). https://doi.org/10.1007/s00216-008-2099-4

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