SNP Genotyping by Unlabeled Probe Melting Analysis

  • Maria Erali
  • Robert Palais
  • Carl Wittwer
Part of the Methods in Molecular Biology book series (MIMB, volume 429)


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 Words

Unlabeled probes genotyping LCGreen Plus asymmetric PCR melting analysis. 


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Copyright information

© Humana Press Inc., Totowa, NJ 2008

Authors and Affiliations

  • Maria Erali
    • 1
  • Robert Palais
    • 2
  • Carl Wittwer
    • 2
  1. 1.Advanced Technology GroupInstitute for Clinical and Experimental Pathology, ARUP LaboratoriesSalt Lake City
  2. 2.Department of MathematicsUniversity of UtahSalt Lake City

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