Improvement of Sensitivity of Molecular Diagnostics by replacing PCR with Cold-PCR

Mike G. Makrigiorgos, Associate Professor, Director Biophysics Laboratory, Dana Farber Cancer Institute

Launch presentation

About the speaker

Dr. Makrigiorgos is the Director of the Medical Physics and Biophysics Division of the combined Radiation Oncology Department at Brigham and Womens, Dana Farber and Children’s Hospitals, and Associate Professor at Harvard Medical School, Boston, USA. He is a Member of the Editorial Board of Clinical Chemistry and has published over 100 articles, reviews and book chapters. His research interests include the development of novel DNA technologies for molecular diagnostics with application in Oncology. He is the inventor of several novel PCR-based techniques for Molecular Diagnostics, including Balanced-PCR, Anti-primer-quenching real time PCR, Hairpin-PCR and COLD-PCR.

Abstract

The use of PCR as the initial step for genetic testing is almost ubiquitous. Here we unveil the strong dependence of PCR amplification efficiency in sequences differing by one or more nucleotides, on the PCR denaturation temperature. Based on this novel PCR property we devised Co-amplification at Lower Denaturation temperature (COLD-PCR, published in Nature Medicine May 2008), a new form of PCR that amplifies minority alleles selectively from mixtures of wild type and mutation-containing sequences irrespective of the mutation type or position on the sequence. , An intermediate temperature is introduced during PCR-cycling to allow cross-hybridization of mutant and wild type alleles; hetero-duplexes are then selectively denatured and amplified at Critical Denaturation Temperature (Tc), while homo-duplexes remain double-stranded and do not amplify efficiently. Di-deoxy-sequencing, pyrosequencing, MALDI-TOF, dHPLC and RFLP improve their sensitivity by up to 100-fold using COLD-PCR. COLD-PCR enables sequencing of mutations in clinical samples and bodily fluids that are not visible using existing approaches and increases the reliability of genetic testing from tumor samples with stromal cell contamination. For micro-deletions the enrichment provided is so pronounced that it can lead to complete isolation of the mutants in a single COLD-PCR reaction. COLD-PCR is expected to have diverse applications in the fields of molecular diagnostics, biomarkers, genomic instability, infectious diseases, methylation testing and pre-natal identification of fetal alleles in mother’s blood, or in any situation where increased mutation detection sensitivity is needed.

Launch presentation