SAN FRANCISCO — Scientists in the laboratory of Mike Makrigiorgos of the Dana Farber Cancer Institute and Harvard Medical School have developed a method that enriches unknown mutations of targeted DNA sequences based purely on thermal denaturation of DNA heteroduplexes without the need for enzymatic reactions.
Using the method, called differential strand separation at critical temperature, or "Dissect," the researchers have been able to perform multiplex enrichment by 100- to 400-fold of KRAS and TP53 mutations, which enabled them to more easily detect these mutations using downstream Sanger sequencing of high-resolution melt analysis.
The technique can also be combined with an earlier innovation from the Makrigiorgos lab — co-amplification at lower denaturation temperature, or Cold-PCR — to detect extremely rare mutations in cancer cells and circulating DNA even when the positions of the mutant sequences are unknown.
Makrigiorgos presented his lab's technology as part of the real-time PCR symposium at Cambridge Healthtech Institute's Molecular Medicine Tri-Conference held here this week.
Makrigiorgos said there is a great need for highly sensitive DNA mutation detection technologies in cancer research, diagnostics, and prognostics, noting that, for example, just one mutation of EGFR T790M among 10,000 wild-type sequences has been shown to cause resistance to tyrosine kinase inhibitors in lung cancers. Low-level mutations such as these are extremely difficult to identify and detect, "especially when their position within the gene is unkwown," Makrigiorgos said.
To address this, the Makrigiorgos lab over the last several years developed the Cold-PCR technique, which takes advantage of the fact that mutant DNA strands denature at lower temperatures in a PCR reaction than normal DNA strands to selectively amplify the mutant DNA with minimal amplification of normal DNA.
The lab has also developed a follow-on technology called improved and complete enrichment, or Ice-Cold-PCR, which enhances this sensitivity by using locked nucleic acid technology and a reference strand that binds PCR amplicons to form duplexes that are preferentially denatured and amplified at a certain temperature.
Both of these methods — as well as several other iterations of the Cold-PCR method — enable researchers to detect low-level mutations without necessarily knowing the location of the mutation. As such, the technology can be combined with downstream detection technologies to identify rare mutations in cancer for the purpose of biomarker and diagnostic development. Omaha, Neb.-based Transgenomic has an exclusive license to analyze Ice-Cold-PCR products using pyro- and Sanger sequencing.
Like Cold-PCR and variations thereof, the Dissect technology uses differential denaturation of DNA heteroduplexes, and as such can enrich mutations at any position on a sequence — mutations that can then be identified using downstream sequencing methods or real-time PCR.
However, a key differentiator of the Dissect technique, Makrigiorgos noted, is that it is based entirely on repeated cycles of hybridization and preferential denaturation on a solid support medium – currently streptavidin-coated magnetic beads.
More specifically, the target DNA is bound to the immobilized oligonucleotides on the beads, and the mixture is heated to a critical temperature, at which point the mutant heteroduplexes are preferentially denatured. Then, the magnetic beads, which are at that point only bound to wild-type DNA, are pulled out of the mixture, leaving only mutant sequences.
This process does not modify the target sequence in any way and can be repeated to produce DNA template enriched for mutations by 200-fold or more.
Makrigiorgos and his colleagues described the method in a paper published in December in Nucleic Acids Research. In that study, they validated Dissect for several DNA mutation targets of clinical interest, such as mutations in KRAS and TP53.
At Tri-Con, Makrigiorgos presented additional data demonstrating how the method could be used to increase the limit of mutation detection by Sanger sequencing and HRM analysis.
He also described initial attempts to multiplex Dissect, enriching for four different mutant DNA targets in a single tube; to use the method to analyze clinical tumor samples and plasma-circulating DNA; and to combine it with Ice-Cold-PCR to further enhance mutation detection.
As described in the Nucleic Acids Research paper, Makrigiorgos and colleagues at first pre-amplified DNA from plasma and clinical samples using 20 cycles of multiplex PCR prior to applying the Dissect technique. However, at Tri-Con, he noted that the group is working on methods to fragment DNA prior to analysis to avoid this pre-amplification step.
Makrigiorgos said that his group has applied for patents on the Dissect technology, but has not yet licensed the technology and is seeking commercial partners.