University of Utah chemists have devised a new way to detect chemical damage to DNA that sometimes leads to genetic mutations responsible for many diseases, including various cancers and neurological disorders.
“We are one step closer to understanding the underlying chemistry that leads to genetic diseases,” says Cynthia Burrows, distinguished professor and chair of chemistry at the university and senior author of a new study published in Nature Communications. “We have a way of marking and copying DNA damage sites so that we can preserve the information of where and what the damage was.”
Jan Riedl, a University of Utah postdoctoral fellow and the study’s first author, says 99 percent of DNA lesions are repaired naturally. The rest can lead to genetic mutations and can cause disease. The new method can “identify and detect the position of lesions that lead to diseases,” he says.
Burrows says: “We are trying to look for the chemical changes in the base that can lead the cell to make a mistake, a mutation. One of the powerful things about our method is we can read more than a single damaged site [and up to dozens] on the same strand of DNA.”
The chemists say their new method will let researchers study chemical details of DNA lesions or damage. Such lesions, if not repaired naturally, accumulate over time and can lead to mutations responsible for many age-related diseases, including neurological ailments such as Huntington’s disease and ALS.
“A method capable of identifying the chemical identity and location in which lesions appear is crucial for determining the molecular etiology [cause] of these diseases,” Burrows and colleague write in their study.
The research was funded by the National Institutes of Health. Burrows and Riedl conducted the study with University of Utah chemists Aaron Fleming, a research assistant professor, and Yun Ding, a postdoctoral researcher.
Method detects DNA damage that can lead to disease
The new method for finding DNA lesions combines other, existing techniques. First, the researchers find the damage and cut it out of the DNA using base excision repair, the discovery of which won a Nobel Prize in Chemistry this year for Tomas Lindahl, a scientist in England.
Second, an “unnatural base pair” is inserted at the snipped-out DNA damage site to label it. Instead of natural base pairs C-G and A-T, the Utah chemists used a so-called third or unnatural base pair invented by chemists at the Scripps Research Institute in California. Burrows says her study demonstrates the first practical use of that invention.
This graphic shows a new method for identifying DNA lesions, or sites of damage on DNA strands that can lead to disease-causing mutations. First the damage is cut out and replaced by so-called unnatural base pairs so chemists can amplify, or make millions of copies, of the damaged DNA. Then they label the unnatural base with a chemical called 18-crown-6 ether. which makes it easier to detect the site of DNA damage by passing the strand of DNA through molecule-size nanopore. Credit: Aaron Fleming / University of Utah
Third, the DNA with the damage site labeled by an unnatural third base pair is then amplified using PCR. Burrows says the new study’s key innovation was to use base excision repair to snip out the damage and then to insert the unnatural base pair at the damage site, making it possible to make millions of copies of the DNA—a process that normally would be prevented by the damage.
Fourth, another chemical label, named 18-crown-6 ether, is affixed to the unnatural base pair on all the DNA strands, which are then read or sequenced using a kind of nanopore sequencing developed a few years ago by Burrows and Utah chemist Henry White. Such sequencing involves determining the order and location of bases on a DNA strand—including damage sites labeled by unnatural bases—by passing the strand through a molecule-size pore or nanopore.
People are born with their genome or genetic blueprint of 3 billion base pairs, “and then stuff happens,” Burrows says. “There’s damage from oxidative stress due to inflammation and infection, too much metabolism, or environmental chemicals.”
The new method seeks “molecular details that define how our genome responds to what we eat and the air we breathe, and ends up being healthy or not,” she says.
DNA lesions happen more than 10,000 times a day in a single human cell. A lesion can be a missing base, a base that has changed chemically or a break in the DNA backbone. That many lesions may seem like a lot, but with 3 billion base pairs in the genome of a single cell, the damage only affects about one of every 300,000 base pairs. The chemists tested their method on a gene named KRAS that, when mutated, can cause lung or breast cancer.
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Riedl, J et al. Identification of DNA lesions using a third base pair for amplification and nanopore sequencing. Nature Communications, Published November 6 2015. doi: 10.1038/ncomms9807