NEW YORK (GenomeWeb News) – An international research team led by investigators in Denmark and Sweden has reportedly developed a new single-molecule DNA barcoding method.
In a paper appearing online recently in the Proceedings of the National Academy of Sciences, the researchers outlined their approach, which relies on moving DNA through nanofluidic channels to distinguish between DNA patterns based on denaturation temperature data. Based on their findings so far, the team says their mapping method may gain favor not only for finding long-range DNA variations — for instance, to distinguish potential pathogens or find telltale changes in the human genome — but also for organizing DNA sequence data.
"The barcode technique could be a simple way to identify what types of virus and bacteria we are dealing with," co-senior author Jonas Tegenfeldt, a solid state physics researcher at Sweden's Lund University, said in a statement. "We can also find out whether something has gone wrong in the human genome, because it is possible to see if any part of the chromosome has moved for any reason."
Because different DNA base pairs are denatured at different temperatures, the researchers explained, it's possible to get a picture of DNA that's stretched into a linear molecule in a nanochannel by labeling it fluorescently and comparing fluorescence signals across the DNA strand.
In general, this means that base pairs that are denatured at lower temperatures, such as adenine and thymine, will show weaker staining than those that are more resilient, such as guanine and cytosine.
"If the DNA is uniformly stained with a dye that unbinds when the DNA melts, local fluorescence of the melted region will decrease," the team noted. "Thus, the partial melting will create a grayscale barcode consisting of brighter and darker regions along the nanochannel-extended molecule."
For their proof-of-principle study, Tegenfeldt and his colleagues developed silica nanochannels in a clean room using electron beam and photo lithography. They then tested their DNA denaturation-based mapping approach using four DNA constructs labeled with the fluorescent stain called YOYO-1, including phage and bacterial artificial chromosome DNA.
Using applied pressure, the team manipulated, moved, and concentrated DNA in the nannochannels. They also relied on a buffer containing the DNA denaturing chemical formamide to lower denaturation temperatures.
The researchers reported that they were, indeed, able to come up with barcodes representing fluorescent highs and lows across the four DNA strands tested. These profiles, in turn, could be compared with fluorescent profiles predicted for each sequence based on melting point information. By barcoding the human chromosome 12, the team showed that the same approach also works for longer pieces of DNA.
The view of the genome remains more imprecise than that possible through genome sequencing. But those involved in the new study say their barcoding method could prove useful for clinical or forensic applications that demand fast detection of sequence variation — and may also serve to help organize DNA sequences obtained using more targeted sequencing strategies.
"[T]he key advantage of the denaturation mapping technique is the ease with which it can detect long-range sequence variation along single DNA molecules," the researchers wrote. "Because the technique does not necessitate DNA fragmentation, large-scale organization of the genome is left intact and observed intact."