KRAS genotyping by digital PCR combined with melting curve analysis

Scientific Reports
Junko TanakaYoshinobu Kohara

Abstract

Digital PCR (dPCR) has been developed as a method that can quantify nucleic acids more sensitively than real-time PCR. However, dPCR exhibits large fluctuations in the fluorescence intensity of the compartment, resulting in low accuracy. The main cause is most likely due to insufficient PCR. In this study, we proposed a new method that combines dPCR with melting curve analysis and applied that method to KRAS genotyping. Since the melting temperature (Tm) of the PCR product hardly depends on the amplification efficiency, genotyping accuracy is improved by using the Tm value. The results showed that the peaks of the distribution of the Tm values of DNA in the wells were 68.7, 66.3, and 62.6 °C for wild-type KRAS, the G12R mutant, and the G12D mutant, respectively, and the standard deviation of the Tm values was 0.2 °C for each genotype. This result indicates that the proposed method is capable of discriminating between the wild-type sequence and the two mutants. To the best of our knowledge, this is the first demonstration of the genotyping of single mutations by combining melting curve analysis and dPCR. The application of this approach could be useful for the quantification and genotyping of cancer-related genes in low-abundanc...Continue Reading

References

Nov 11, 1990·Nucleic Acids Research·W RychlikR E Rhoads
Oct 1, 1996·Genome Research·C A HeidP M Williams
Jul 15, 2003·Proceedings of the National Academy of Sciences of the United States of America·Devin DressmanBert Vogelstein
Jun 7, 2005·Proceedings of the National Academy of Sciences of the United States of America·Kenneth E PierceLawrence J Wangh
Jun 23, 2006·Nature Methods·Frank DiehlDevin Dressman
Nov 14, 2006·Proceedings of the National Academy of Sciences of the United States of America·Luigi WarrenStephen R Quake
Dec 2, 2006·Science·Elizabeth A OttesenJared R Leadbetter
Aug 13, 2008·Methods in Molecular Biology·Ulf B Christensen
Jan 28, 2009·Journal of Clinical Microbiology·Hiyam H El-HajjDavid Alland
May 1, 2010·Scandinavian Journal of Infectious Diseases·Vivian KjellandAudun Slettan
May 13, 2011·Nature Reviews. Cancer·Heidi SchwarzenbachKlaus Pantel
Nov 1, 2011·Analytical Chemistry·Benjamin J HindsonBill W Colston
Nov 5, 2013·Analytical Chemistry·Geoffrey P McDermottAdam J Lowe
Apr 10, 2015·Nature Communications·Agnieszka K WitkiewiczErik S Knudsen
Dec 17, 2015·Scientific Reports·Erina TakaiShinichi Yachida
Mar 13, 2016·Cancer Discovery·Catherine Alix-Panabières, Klaus Pantel
Mar 25, 2016·Clinical and Translational Gastroenterology·Barbara BournetLouis Buscail

❮ Previous
Next ❯

Citations

Sep 19, 2020·Analytical Chemistry·Ahmad MoniriJesus Rodriguez-Manzano

❮ Previous
Next ❯

Methods Mentioned

BETA
biopsy
PCR
flow cytometry
chip
genotyping
Fluorescence

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.

Related Papers

Journal of Clinical Microbiology
Jane Kuypers, Keith R Jerome
Bing du xue bao = Chinese journal of virology
Zhaomin Feng, Yuelong Shu
Water Science and Technology : a Journal of the International Association on Water Pollution Research
Naohiro KishidaYuji Sekiguchi
© 2022 Meta ULC. All rights reserved