Detection of low-level mutations in DNA obtained from cancer patients, using PCR and nano-PCR
Mentor: Mike Makrigiorgos, PhD (Dana Farber Cancer Institute)
Screening of individual patients’ tumors for genetic alterations in cancer-related genes in a rapid and cost-effective manner is a significant challenge and a requirement that must be fulfilled in order to realize the promise of individualized cancer treatment. Mutations in multiple genes along signaling pathways that govern cell cycle, growth, apoptosis and genome integrity are critical for early cancer detection, prediction of the pathologic behavior of malignancies and responsiveness of particular tumors to treatment. Low-level tumor somatic DNA mutations, in particular, can have profound implications for development of metastasis, prognosis, choice of treatment, follow-up or early cancer detection. Unless effectively detected, these low-level mutations can misinform patient management decisions or become missed opportunities for personalized medicine. And yet, the clinical significance of identifying these low-level somatic mutations is major. For example, mutations in plasma and liquid biopsies from cancer patients can be useful as biomarkers for early cancer detection or for monitoring tumor response to treatment. Unfortunately, such low-level mutations can be ‘masked’ by presence of wild type alleles, and their detection is technically difficult. This project will utilize novel PCR-based approaches to enable detection of low-level mutations via mutation enrichment. In particular, co-amplification at lower denaturation temperature (COLD-PCR) is a PCR-based method that suppresses amplification of wild-type alleles during PCR. COLD-PCR will be adapted to operate in nano-liter-sized volumes such that, multiple nano-reactions addressing different DNA targets of clinical significance take place simultaneously. Thereby amplifying preferentially, the mutated DNA alleles in multiplexed manner and facilitating their detection even at very low mutant-allele frequencies (MAF). Similarly, enzymatic approaches to degrade selectively wild-type DNA using double-strand DNA-specific nuclease will be adopted, either alone or in combination with nano-PCR. These approaches will be applied in mutated cell lines and clinical samples obtained from cancer patients to reveal cancer-relevant mutations that help with management decisions.
Various applications of PCR for use in molecular diagnostics Source: