DNA mutations can cause many diseases such as cancer. But by the time scientists identify DNA mutations, the original chemical damage that caused them is lost.
“What we’d really like to do is sequence for the chemical modifications, the origins of the mutations,” says Cynthia J. Burrows, a chemistry professor at the University of Utah.
Unfortunately, damaged nucleotides block the enzymes needed to amplify DNA for sequencing. Burrows and coworkers have overcome this barrier with a method that amplifies and detects DNA lesions. To do that, they mark DNA damage with an unnatural base pair that is compatible with the polymerase chain reaction, or PCR (Nat. Commun. 2015, DOI: 10.1038/ncomms9807).
First they use enzymes from the DNA base excision repair pathway to identify and clip out the damage, leaving a gap in the DNA. On the basis of which enzymes they end up needing, the team can determine the kind of damage that was present.
Next, Burrows and coworkers fill the gap with hydrophobic unnatural bases developed by Floyd Romesberg at Scripps Research Institute, La Jolla, Calif. “We just give it one nucleotide in relatively high concentration and a polymerase enzyme,” Burrows says. Such conditions force the polymerase to incorporate the unnatural nucleotide, even though it’s a mismatch with the base sitting across from the gap.
Then the researchers amplify the mismatched duplex using PCR. By including a second unnatural nucleotide that is fully complementary to first, they are able to generate many copies of DNA with the unnatural base pair.
Biochemists previously showed that polymerases could incorporate hydrophobic nucleotides across from gaps in a DNA sequence, says Eric T. Kool, a chemistry professor at Stanford University who studies unnatural nucleotides. “However, the new work takes an important practical step ahead by employing a full base pair that can be amplified by PCR.”
Finally, the Utah team takes those DNA copies and uses protein nanopore sequencing to identify the location of the unnatural base, which tells them where the lesion was originally. To make the unnatural bases easy to detect, they label them with a crown ether that modulates the current measured through the nanopore.
“The diversity of lesion structures and their exceedingly low abundance in the genome makes it seem like an impossible problem” to spot and characterize them, says Shana J. Sturla of ETH Zurich, who previously developed a method for marking a particular type of lesion. “By developing a clever strategy that brings together different enzymological and chemical tricks, whereby the lesion can be found, marked, amplified, and detected, Burrows and coworkers start to make the impossible seem possible.”