The chemists report the advance in the week of June 18 online edition of the journal Proceedings of the National Academy of Sciences.
“We’re using this technique and synthetic organic chemistry to be able to see a damage site as it flies through the nanopore,” says Henry White, distinguished professor and chair of chemistry at the University of Utah and senior coauthor of the new study.
Strands of DNA are made of “nucleotide bases” known as A, T, G and C. Some stretches of DNA strands are genes.
The new method looks for places where a base is missing, known as an “abasic site,” one of the most frequent forms of damage in the 3-billion-base human genome or genetic blueprint. This kind of DNA damage happens 18,000 times a day in a typical cell as we are exposed to everything from sunlight to car exhaust. Most of the damage is repaired, but sometimes it leads to a gene mutation and ultimately disease.
By combining nanopore damage-detection with other chemical ways of altering DNA, the researchers hope to make this new technique capable of detecting other kinds of DNA damage by converting the damage to a missing base, says the study’s other senior coauthor, Cynthia Burrows, a distinguished professor of chemistry at the University of Utah.She adds: “Damage to the bases of DNA contributes to many age-related diseases, including melanoma; lung, colon and breast cancers; Huntington’s disease; and atherosclerosis.”
White and Burrows conducted the study with first author, Na An, a doctoral student in chemistry and Aaron Fleming, a postdoctoral research associate in chemistry. The study was funded by the National Institutes of Health, with equipment and software donations by Electronic BioSciences of San Diego.
Toward Cheaper, Faster DNA Sequencing
Sequencing is the process of determining the order of the nucleotide bases A, C, G and T in one of the two strands of bases that make up a DNA double helix. It is the basic method used to determine the genomes, or genetic blueprints, of living organisms and to identify disease-causing mutations in genes.“Twenty years ago, it cost $1 billion to sequence the first human genome,” while the cost now runs from $5,000 to $20,000, White says. “The National Institutes of Health has had the $1,000 genome project for a few years, and the price likely will go lower.”
Nanopore sequencing is performed by passing a strand of DNA through a nanoscopic pore while both are bathed in an electrically charged solution known as an electrolyte. Some of that solution also is flowing through the pore. Researchers detect different current levels as differing DNA bases pass through the pore, blocking varying amounts of the electrified solution from passing through the pore.
Using Nanopores to Look for Damage
Unlike efforts to achieve nanopore sequencing of DNA, the Utah chemists are not reading the sequence of DNA bases as the strand move through a pore – although they eventually want to do so – but “we are detecting single base damage,” White says.
“It’s important to know how a damaged base leads to a mutation because that is the first step in a disease occurring,” he adds. “Right now, we can see the damaged site and tell approximately where it is within the piece of DNA we’re analyzing” – to within about five or 10 bases. The goal is to pinpoint damage sites, and to understand how damage at specific sites leads to disease.
So far, the longest piece of DNA the Utah chemists put through a nanopore was about 100 bases long, and they were able to detect one or two damage sites.“We’ve still got to do a lot of research and come up with ways of improving this,” White says. “It’s a very promising and new way of doing it. There are no other ways of doing what we’re trying to do,” namely, not only identify damage, but get sequence information to pinpoint damaged locations on a DNA strand.
The researchers created damage on some DNA by removing some bases. Where bases were missing, the sugar in the DNA backbone was exposed. The chemists attached a ring- or crown-shaped chemical known as an “18-crown-6 ether” to the sugar.The trick was to get the DNA, with the crown ether attached, to pass through the nanopore slowly enough so missing bases can be detected.
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