Abstract
A number of molecular diagnostic assays have been developed in the last years for mutation detection. Although these methods have become increasingly sensitive, most of them are incompatible with a sequencing-based readout and require prior knowledge of the mutation present in the sample. Consequently, coamplification at low denaturation (COLD)-PCR-based methods have been developed and combine a high analytical sensitivity due to mutation enrichment in the sample with the identification of known or unknown mutations by downstream sequencing experiments. Among these methods, the recently developed Enhanced-ice-COLD-PCR appeared as the most powerful method as it outperformed the other COLD-PCR-based methods in terms of the mutation enrichment and due to the simplicity of the experimental setup of the assay. Indeed, E-ice-COLD-PCR is very versatile as it can be used on all types of PCR platforms and is applicable to different types of samples including fresh frozen, FFPE, and plasma samples. The technique relies on the incorporation of an LNA containing blocker probe in the PCR reaction followed by selective heteroduplex denaturation enabling amplification of the mutant allele while amplification of the wild-type allele is prevented. Combined with Pyrosequencing®, which is a very quantitative high-resolution sequencing technology, E-ice-COLD-PCR can detect and identify mutations with a limit of detection down to 0.01 %.
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References
Wittwer CT (2009) High-resolution DNA melting analysis: advancements and limitations. Hum Mutat 30:857–859
Ugozzoli L, Wallace RB (1991) Allele-specific polymerase chain reaction. Methods 2:42–48
Ogino S, Kawasaki T, Brahmandam M et al (2005) Sensitive sequencing method for KRAS mutation detection by Pyrosequencing. J Mol Diagn 7:413–421
Ahmadian A, Ehn M, Hober S (2006) Pyrosequencing: history, biochemistry and future. Clin Chim Acta 363:83–94
Ronaghi M, Shokralla S, Gharizadeh B (2007) Pyrosequencing for discovery and analysis of DNA sequence variations. Pharmacogenomics 8:1437–1441
Ronaghi M, Uhlen M, Nyren P (1998) A sequencing method based on real-time pyrophosphate. Science 281:363–365
Ronaghi M (2001) Pyrosequencing sheds light on DNA sequencing. Genome Res 11:3–11
Milbury CA, Li J, Makrigiorgos GM (2009) PCR-based methods for the enrichment of minority alleles and mutations. Clin Chem 55:632–640
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. Nat Med 14:579–584
Milbury CA, Li J, Makrigiorgos GM (2009) COLD-PCR-enhanced high-resolution melting enables rapid and selective identification of low-level unknown mutations. Clin Chem 55:2130–2143
Milbury CA, Li J, Makrigiorgos GM (2011) Ice-COLD-PCR enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations. Nucleic Acids Res 39:e2
How Kit A, Mazaleyrat N, Daunay A et al (2013) Sensitive detection of KRAS mutations using enhanced-ice-COLD-PCR mutation enrichment and direct sequence identification. Hum Mutat 34:1568–1580
Koressaar T, Remm M (2007) Enhancements and modifications of primer design program Primer3. Bioinformatics 23:1289–1291
Untergasser A, Cutcutache I, Koressaar T et al (2012) Primer3—new capabilities and interfaces. Nucleic Acids Res 40:e115
Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415
Forbes SA, Bindal N, Bamford S et al (2011) COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic Acids Res 39:D945–D950
Dunker J, Larsson U, Petersson D et al (2003) Parallel DNA template preparation using a vacuum filtration sample transfer device. Biotechniques 34:862–868
Zhou GH, Gotou M, Kajiyama T et al (2005) Multiplex SNP typing by bioluminometric assay coupled with terminator incorporation (BATI). Nucleic Acids Res 33:e133
You Y, Moreira BG, Behlke MA et al (2006) Design of LNA probes that improve mismatch discrimination. Nucleic Acids Res 34:e60
Acknowledgments
We thank Florence Mauger (CNG), Helene Myrtue Nielsen (Aarhus University), and Steven McGinn (CNG) for critical reading and improvement of the manuscript.
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How-Kit, A., Tost, J. (2015). Pyrosequencing®-Based Identification of Low-Frequency Mutations Enriched Through Enhanced-ice-COLD-PCR. In: Lehmann, U., Tost, J. (eds) Pyrosequencing. Methods in Molecular Biology, vol 1315. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2715-9_7
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DOI: https://doi.org/10.1007/978-1-4939-2715-9_7
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