DNA molecules move in mysterious ways

The copolymer mainly consists of linear polyacrylamide (LPA), which is already commonly used for separating DNA in microchip capillary electrophoresis (CE), combined with much smaller amounts of dihexyacrylamide (DHA). The hydrophobic (water-hating) DHA molecules form outcrops on the hydrophilic (water-loving) LPA strands, with around 20 DHA molecules spread over four or five polymer blocks. The researchers, led by Annelise Barron from Northwestern University, Illinois, found that this LPA-co-DHA copolymer would form physical cross-links in solution, generating a solid gel. These cross-links form because the DHA molecules naturally bunch together on exposure to water, causing the individual copolymer strands to become tangled up. Barron and her colleagues had previously developed this polymer to purify DNA samples by removing proteins, but they guessed that it would also prove effective at separating different size DNA strands in microchip CE. So they tested its ability to separate the various different length DNA strands that make up DNA ladders. They found that the LPA-co-DHA copolymer was more effective than standard LPA at separating the different size DNA strands, generating clear, well-separated peaks in the subsequent electropherogram. Indeed, the LPA-co-DHA copolymer was sensitive enough to detect minor irregularities in the DNA ladders. The researchers found that certain of the peaks generated by the LPA-co-DHA copolymer contained one or more sub-peaks. Further research showed that this was because some supposedly identical strands in the DNA ladder actually varied in size by a few base pairs, due to the difficulty of synthesising DNA strands of exact sizes. For instance, one of the DNA ladders contained strands from 50 to 800 base pairs (bp) in length, increasing by 50 bp intervals. However, the researchers found that, along with the 350 bp strands, the DNA ladder also contained a few 360 bp strands. This slight size difference was not picked up when separating the ladder using LPA, which simply generated a single peak, but was picked up by the LPA-co-DHA copolymer. To investigate the copolymer's performance in more detail, Barron and her team monitored the migration of individual DNA strands using single-molecule epifluorescent videomicroscopy. This instrument effectively consists of a powerful microscope hooked up to a CCD (charge-coupled device) camera and was specifically developed to monitor DNA migration in CE. The researchers first investigated the movement of DNA molecules in standard LPA and recorded some of the first images of the two best-known DNA migration mechanisms. In low concentration LPA gels, DNA molecules migrate via a mechanism known as transient entanglement coupling. This involves a DNA molecule becoming hooked around successive polymer strands, and pulling them through the gel solution before breaking free. In high concentration LPA gels, in contrast, DNA molecules migrate via a mechanism known as reptation. This involves the molecules effectively squeezing through tiny tunnels between the packed-together polymer strands. In the LPA-co-DHA copolymer, however, Barron and her colleagues recorded an entirely novel migration mechanism, which they termed stationary entanglement coupling. Like transient entanglement coupling, the DNA molecule becomes hooked around successive polymer strands, but it is unable to pull the strands through the gel solution and simply has to move around them to break free. The researchers think that this novel mechanism helps to explain the superior separation ability of the LPA-co-DHA copolymer. Barron and her colleagues are now developing this copolymer for a variety of applications, including forensics, gene sequencing and DNA purification. "We are talking with several companies interested in licensing the patents and sponsoring further development - Agilent, Microchip Biotechnologies and Network Biosystems," she told separationsNOW, "and I am also in the process of starting my own company to commercialize these and other novel polymer networks for integrated bioanalytical chip devices." "Source":[ http://www.separationsnow.com/coi/cda/detail.cda?id=14045&type=Feature&chId=2&page=1]

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