NEW YORK (GenomeWeb) – A team of researchers from the University of Utah has developed a new method to find DNA damage using third base pair labeling and nanopore sequencing.
The method could help scientists better understand how such DNA damage leads to specific mutations underlying a number of diseases, including various cancers and neurological disorders. The work also demonstrates the potential of nanopore-based sequencing to detect non-native nucleotides, previously only achievable using other sequencing methods.
Damage to genomes, in the form of lesions, is inherent in stress-induced disease and cancer progression. However, these lesions exist in low levels that make it impossible to amplify by standard PCR methods.
As they described in a paper published in Nature Communications last week, the Utah researchers developed a four-step reaction sequence followed by PCR amplification and sequencing.
"The critical issue is that damaged DNA [normally] doesn’t get amplified," said Cynthia Burrows, chemist at the University of Utah and lead author on the study. Burrows and her colleagues decided to use the third base pair, originally developed by Floyd Romesberg from Scripps Research Institute, to label areas with damaged DNA because it goes through PCR amplification very nicely, but is also easily differentiated from A, C, G, and T base pairs.
To label the damaged DNA, the researchers installed either dNaM or d5SICS nucleotides at the lesion site. These marker nucleotides constitute an unnatural third base pair, allowing large quantities of marked DNA to be made using New England Biolabs’ OneTaq DNA Polymerase for PCR.
The method builds upon a previous technique developed by the same Utah researchers that involved a chemical modification method for marking lesion sites as DNA moved through a nanopore system. Specifically, they focused on sites that were missing a purine or pyrimidine base in order to find those DNA damage sites. This type of chemical modification takes advantage of the chemical properties at abasic sites. Using this method, the Utah researchers were able to determine what type of electrical signal is produced at these types of damaged DNA sites.
Researchers established the new method’s efficacy by using Thermo Fisher Scientific's BigDye Terminator v3.1 Cycle Sequencing kit to perform Sanger sequencing on a section of the KRAS gene in codon 12. The researcher chose to look at the KRAS gene site because when it mutates it often leads to the development of lung and colon cancer, said Burrows.
However, to analyze the markers in individual DNA strands Burrows and her colleagues decided to do nanopore sequencing, specifically α-hemolysin nanopore analyses.
"It turned out that it was particularly useful to use nanopore sequencing technology because that allowed us to look at multiple sites in the strand," Burrows told GenomeWeb. "We want to look not at just one site, but all the sites that have undergone damage to the base."
The researchers believe that the method could be used to help answer questions about the underlying chemical reactions that initiate mutations in the genome that lead to disease. For example, mutations in codon 12 of the KRAS gene can be probed to determine if dOG or dU — common deaminiation and oxidative lesions — are responsible for the mutations observed in lung or colon cancer, respectively. They also stated in the paper that being able to better identify DNA damage and the potential for mutation will be paramount in the development of preventative medicine for cancers and other deleterious stress-induced diseases resulting from these mutations.
The researchers also noted that the new α-HL nanopore sequencing methodology they developed has the potential to become a robust approach for detecting and sequencing non-native nucleotides. Currently, detecting and sequencing non-native nucleotides is only achievable by single-molecule real-time sequencing, Burrows noted that it would be possible to use other sequencing methods to analyze DNA damage sites, but it would require the development of dye labels for methods such as Illumina sequencing.