Label-free single-molecule DNA sequencing
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microfluidics can be used to trap a single DNA–enzyme complex in its native state, without immobilisation. These studies have usually been performed by immobilising either the enzyme or the DNA on a glass slide, but this may modify their properties, and make it difficult to analyse the products.
This technique gives information about how enzymes that are used to modify DNA, called restriction enzymes, act and holds promise for a broad range of DNA–protein interaction studies.
Restriction enzymes are tools used by molecular biologosts to chop up DNA into pieces, making sequencing possible. The enzymes work by binding to a particular DNA sequence, and then cutting the DNA at that point. However, determining the enzymes’ mechanism of action is difficult, something that is now being addressed by using single-molecule studies. These have proved to be a powerful tool, as they avoid the problem of properties being averaged over a population, and you can follow complex processes in real-time.
An enzyme from E. coli was pre-bound to bacteriophage DNA and fed through a microfluidic system. This trapped the complex, and then stretched it out at a stagnation point. Adding Mg2+ then activated the enzyme, cleaving the DNA, and permitting analysis of the products. The accuracy in determining the location of the recognition site is comparable to or better than other single-molecule techniques.
Original publication: W Xu and S J Muller, Lab Chip, 2010.
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