Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing

Nature Medicine
Jin LiG Mike Makrigiorgos

Abstract

PCR is widely employed as the initial DNA amplification step for genetic testing. However, a key limitation of PCR-based methods is the inability to selectively amplify low levels of mutations in a wild-type background. As a result, downstream assays are limited in their ability to identify subtle genetic changes that can have a profound impact in clinical decision-making and outcome. Here we describe co-amplification at lower denaturation temperature PCR (COLD-PCR), a novel 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. We replaced regular PCR with COLD-PCR before sequencing or genotyping assays to improve mutation detection sensitivity by up to 100-fold and identified new mutations in the genes encoding p53, KRAS and epidermal growth factor in heterogeneous cancer samples that had been missed by the currently used methods. For clinically relevant microdeletions, COLD-PCR enabled exclusive amplification and isolation of the mutants. COLD-PCR will transform the capabilities of PCR-based genetic testing, including applications in cancer, infectious diseases and prenatal identification of fetal alle...Continue Reading

References

Aug 16, 1997·Lancet·Y M LoJ S Wainscoat
Nov 26, 1998·Journal of Clinical Pathology·F MayallB Chang
Nov 4, 2000·Annals of Oncology : Official Journal of the European Society for Medical Oncology·R GonzalezF Bonilla
Feb 1, 2002·Nature Biotechnology·Xiyuan SunBaochuan Guo
Jul 15, 2004·Annals of the New York Academy of Sciences·Tatsuo KimuraPaul H Gumerlock
Feb 25, 2005·The New England Journal of Medicine·Susumu KobayashiBalázs Halmos
Jun 14, 2005·BioTechniques·Anthony T YeungEmmanuelle Nicolas
Jul 29, 2005·The Journal of Molecular Diagnostics : JMD·Shuji OginoCharles S Fuchs
Sep 17, 2005·Clinical Cancer Research : an Official Journal of the American Association for Cancer Research·Steven A BelinskyMaria A Picchi
Nov 1, 2005·Proceedings of the National Academy of Sciences of the United States of America·Frank DiehlBert Vogelstein
Feb 10, 2006·Clinical Cancer Research : an Official Journal of the American Association for Cancer Research·Pasi A JänneBruce E Johnson
Aug 15, 2006·The Journal of Clinical Investigation·Jeffrey A EngelmanPasi A Jänne
Feb 13, 2007·Nature Genetics·Roman K ThomasLevi A Garraway
Mar 9, 2007·Nature·Christopher GreenmanMichael R Stratton
Jun 20, 2007·Nucleic Acids Research·Christian HoffmannFrederic D Bushman

❮ Previous
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Citations

Jul 31, 2013·Current Hematologic Malignancy Reports·Robert J Kreitman
May 12, 2009·Modern Pathology : an Official Journal of the United States and Canadian Academy of Pathology, Inc·Zhuang ZuoRajyalakshmi Luthra
May 25, 2013·Modern Pathology : an Official Journal of the United States and Canadian Academy of Pathology, Inc·Jerzy LasotaMarkku Miettinen
May 19, 2011·Proceedings of the National Academy of Sciences of the United States of America·Isaac KindeBert Vogelstein
Oct 13, 2010·Nucleic Acids Research·Coren A MilburyG Mike Makrigiorgos
Jul 19, 2012·International Journal of Clinical Practice·A PennycuickJ Spicer
Jun 1, 2012·Science Translational Medicine·Tim ForshewNitzan Rosenfeld
May 25, 2011·BMC Plant Biology·Yihua Chen, H Dayton Wilde
Oct 12, 2010·Journal of Experimental & Clinical Cancer Research : CR·Gillian EllisonGeorgina Speake
Dec 5, 2012·Hereditary Cancer in Clinical Practice·Grzegorz KurzawskiRodney J Scott
Jan 1, 2008·Hereditary Cancer in Clinical Practice·Joanna MatyjasikGrzegorz Kurzawski
Dec 14, 2012·PloS One·Elena Castellanos-RizaldosG Mike Makrigiorgos
Feb 10, 2009·Clinical Chemistry·Coren A MilburyG Mike Makrigiorgos
Oct 27, 2010·Clinical Chemistry·Silvia GalbiatiLaura Cremonesi
Dec 24, 2011·Clinical Chemistry·Coren A MilburyG Mike Makrigiorgos
May 17, 2012·Clinical Chemistry·E Castellanos-RizaldosG Mike Makrigiorgos
Jan 1, 2011·Pharmacogenomics and Personalized Medicine·Arun P Wiita, Iris Schrijver
May 19, 2010·Biomarkers in Medicine·Barbara L Parsons, Fanxue Meng
Jun 23, 2011·Pharmacogenomics·Jean-Étienne MorlighemAlexander Lezhava
May 17, 2014·European Journal of Cancer : Official Journal for European Organization for Research and Treatment of Cancer (EORTC) [and] European Association for Cancer Research (EACR)·Virginia Alonso-EspinacoJoan Maurel
Aug 12, 2014·Journal of the National Cancer Institute·Jason J LukeUNKNOWN Cell Free DNA Working Group
Oct 5, 2010·Annual Review of Pathology·Gilda da Cunha SantosMing Sound Tsao
Nov 5, 2014·The Journal of Chemical Physics·Karthikeyan Marimuthu, Raj Chakrabarti
Nov 22, 2015·Expert Review of Molecular Diagnostics·Jörg Tost

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