Temperature-tolerant COLD-PCR reduces temperature stringency and enables robust mutation enrichment.

Clinical Chemistry
Elena Castellanos-RizaldosG Mike Makrigiorgos

Abstract

Low-level mutations in clinical tumor samples often reside below mutation detection limits, thus leading to false negatives that may impact clinical diagnosis and patient management. COLD-PCR (coamplification at lower denaturation temperature PCR) is a technology that magnifies unknown mutations during PCR, thus enabling downstream mutation detection. However, a practical difficulty in applying COLD-PCR has been the requirement for strict control of the denaturation temperature for a given sequence, to within ±0.3 °C. This requirement precludes simultaneous mutation enrichment in sequences of substantially different melting temperature (T(m)) and limits the technique to a single sequence at a time. We present a temperature-tolerant (TT) approach (TT-COLD-PCR) that reduces this obstacle. We describe thermocycling programs featuring a gradual increase of the denaturation temperature during COLD-PCR. This approach enabled enrichment of mutations when the cycling achieves the appropriate critical denaturation temperature of each DNA amplicon that is being amplified. Validation was provided for KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) and TP53 (tumor protein p53) exons 6-9 by use of dilutions of mutated DNA, clini...Continue Reading

References

May 27, 2003·Clinical Chemistry·Carl T WittwerRobert J Pryor
Feb 10, 2009·Clinical Chemistry·Coren A MilburyG Mike Makrigiorgos
Mar 17, 2009·Modern Pathology : an Official Journal of the United States and Canadian Academy of Pathology, Inc·David DelaneyAdrienne M Flanagan
Mar 18, 2009·Biochemical Society Transactions·Jin Li, G Mike Makrigiorgos
May 12, 2009·Modern Pathology : an Official Journal of the United States and Canadian Academy of Pathology, Inc·Zhuang ZuoRajyalakshmi Luthra
Nov 3, 2009·Nature Biotechnology·Ryan TewheyKelly A Frazer
Oct 5, 2010·Human Mutation·Blandine BoisselierMarc Sanson
Oct 13, 2010·Nucleic Acids Research·Coren A MilburyG Mike Makrigiorgos
Oct 27, 2010·Clinical Chemistry·Silvia GalbiatiLaura Cremonesi
Jan 26, 2011·Clinica Chimica Acta; International Journal of Clinical Chemistry·Pamela PinzaniClaudio Orlando
Mar 17, 2011·Expert Review of Molecular Diagnostics·Coren A MilburyG Mike Makrigiorgos
Apr 19, 2011·The Journal of Molecular Diagnostics : JMD·Maurice ChanAnn Siew Gek Lee
May 3, 2011·Diagnostic Molecular Pathology : the American Journal of Surgical Pathology, Part B·Chen SongGerasimos Mike Makrigiorgos
May 25, 2011·BMC Plant Biology·Yihua Chen, H Dayton Wilde
Oct 6, 2011·International Journal of Oncology·Pietro CarotenutoNicola Normanno
Dec 24, 2011·Clinical Chemistry·Coren A MilburyG Mike Makrigiorgos
Aug 28, 2012·Oncology Reports·Yasuhiro InoueMasato Kusunoki

❮ Previous
Next ❯

Citations

Dec 14, 2012·PloS One·Elena Castellanos-RizaldosG Mike Makrigiorgos
Mar 17, 2015·The Journal of Molecular Diagnostics : JMD·Elena Castellanos-RizaldosG Mike Makrigiorgos
Feb 2, 2017·International Journal of Molecular Sciences·Julie A VendrellJérôme Solassol
Jan 20, 2017·Molecular Diagnosis & Therapy·Florence MaugerJörg Tost
Jan 17, 2019·Expert Review of Molecular Diagnostics·Claire FranczakAlexandre Harlé
Oct 10, 2014·Clinical Chemistry·Elena Castellanos-RizaldosMike G Makrigiorgos
May 7, 2014·Clinical Chemistry·Derek M MurphyG Mike Makrigiorgos
Jun 24, 2016·Clinical Chemistry·Florence MaugerAlexandre How-Kit
Dec 12, 2020·Expert Review of Molecular Diagnostics·Andrea Moreno-ManuelEloisa Jantus-Lewintre
Nov 16, 2014·Clinical Cancer Research : an Official Journal of the American Association for Cancer Research·Athina MarkouEvi Lianidou

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.