Advantages of touchdown pcr9/11/2023 ![]() Zuo Z, Chen SS, Chandra PK et al (2009) Application of COLD-PCR for improved detection of KRAS mutations in clinical samples. Appl Immunohistochem Mol Morphol 22(2):114–118 Mairinger FD, Vollbrecht C, Streubel A et al (2014) The “COLD-PCR approach” for early and cost-effective detection of tyrosine kinase inhibitor resistance mutations in EGFR-positive non-small cell lung cancer. In the touchdown PCR, By sequentially decreasing the annealing temperature during each PCR cycle, the chance of the non-specific binding can be reduced. Milbury CA, Li J, Makrigiorgos GM (2009) COLD-PCR-enhanced high-resolution melting enables rapid and selective identification of low-level unknown mutations. Li J, Wang L, Mamon H et al (2008) Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Lin SY, Su YN, Hung CC et al (2008) Mutation spectrum of 122 hemophilia A families from Taiwanese population by LD-PCR, DHPLC, multiplex PCR and evaluating the clinical application of HRM. Nomoto K, Tsuta K, Takano T et al (2006) Detection of EGFR mutations in archived cytologic specimens of non-small cell lung cancer using high-resolution melting analysis. Li J, Wang L, Janne PA, Makrigiorgos GM (2009) Coamplification at lower denaturation temperature-PCR increases mutation-detection selectivity of TaqMan-based real-time PCR. Yamamoto M, Kakihana K, Ohashi K et al (2009) Serial monitoring of T315I BCR-ABL mutation by Invader assay combined with RT-PCR. Ogino S, Kawasaki T, Brahmandam M et al (2005) Sensitive sequencing method for KRAS mutation detection by pyrosequencing. Luthra R, Zuo Z (2009) COLD-PCR finds hot application in mutation analysis. Li J, Makrigiorgos GM (2009) COLD-PCR: a new platform for highly improved mutation detection in cancer and genetic testing. Kobayashi S, Boggon TJ, Dayaram T et al (2005) EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. Lo YM, Corbetta N, Chamberlain PF et al (1997) Presence of fetal DNA in maternal plasma and serum. Co-amplification at lower denaturation temperature PCR (COLD-PCR).We describe in this chapter our COLD-PCR-based pyrosequencing method for KRAS mutation detection in various clinical samples using DNA extracted from either fresh or fixed paraffin-embedded tissue specimens. COLD-PCR can be a good strategy for mutation detection in specimens with high nonneoplastic cell content, small specimens in which neoplastic cells are difficult to micro-dissect and therefore enrich, and whenever a mutation is suspected to be present but is undetectable using conventional PCR and sequencing methods. This method is based on exploitation of the critical temperature, Tc, at which mutation-containing DNA is preferentially melted over wild type. Co-amplification at lower denaturation temperature-based polymerase chain reaction (COLD-PCR) is a single-step amplification method that results in the enhancement of both known and unknown minority alleles during PCR, irrespective of mutation type and position.
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