SNP Genotyping by Unlabeled Probe Melting Analysis
Fluorescent nucleic acid detection in polymerase chain reaction (PCR) generally uses oligonucleotide probes labeled with covalently attached dyes. However, unlabeled oligonucleotides in the presence of saturating DNA dyes can also serve as hybridization probes. The DNA dye, LCGreen® Plus, and a 3′-blocked unlabeled probe are added before amplification, and asymmetric PCR is performed at a 1:5 to 1:10 primer ratio. After PCR is complete, fluorescent melting curves reveal both probe melting at low temperature and amplicon melting at high temperature. After background removal, the melting temperature(s) of the probe/target duplex specific to the allele(s) amplified are revealed. Probes between 20 and 40 bp with T ms between 50 and 85°C are effective. The method requires only three standard oligonucleotides and endpoint fluorescence melting. No real-time PCR or allele-specific amplification is needed. Unlabeled probes are inexpensive, provide the sequence specificity of probes, and allow simultaneous identification of multiple alleles by melting analysis.
Key WordsUnlabeled probes genotyping LCGreen Plus asymmetric PCR melting analysis.
- 5.Prince, J. A., Feuk, L., Howell, W. M., Jobs, M., Emahazion, T., Blennow, K., and Brookes, A. J. (2001) Robust and accurate single nucleotide polymorphism genotyping by dynamic allele-specific hybridization (DASH): design criteria and assay validation. Genome Res. 11, 152–162.CrossRefPubMedGoogle Scholar
- 8.Wittwer, C. T., Dujols, V. E., Reed, G., and Zhou, L. (2004) Amplicon melting analysis with saturation dyes. US Patent Application 2006-0019253 A1.Google Scholar
- 10.Wittwer, C. T. and Kusukawa, N. (2004) Real-time PCR. Pages 71–84 in D. H. Persing, Tenover, F. C., Versalovic, J., Tang, Y. W., Unger, E. R., Relman, D. A., and White, T. J., eds. Diagnostic molecular microbiology; principles and applications. ASM Press, Washington DC.Google Scholar
- 14.Cradic, K. W., Wells, J. E., Allen, L., Kruckeberg, K. E., Singh, R. J., and Grebe, S. K. (2004) Substitution of 3′-phosphate cap with a carbon-based blocker reduces the possibility of fluorescence resonance energy transfer probe failure in real-time PCR assays. Clin. Chem. 50, 1080–1082.CrossRefPubMedGoogle Scholar