Zip nucleic acid: a new reliable method to increase the melting temperature of real-time PCR probes

Abstract

TaqMan genotyping with real-time PCR is a reliable method for single nucleotide polymorphism detection, which is done by probes. These oligonucleotides should be short enough to avoid mismatch hybridization, as well as having 5-10°C higher melting temperature than the primers of real-time PCR reaction. One approach for these qualities is to conjugate the probe with minor groove binder (MGB). Having no access to MGB probes, we searched for an alternative. In the current study, we used Zip Nucleic Acids (ZNA) as probes to increase its stability and melting temperature. Our aim was to genotype the -265 T/C changes of Apolipoprotein A-2 gene. We set up the real-time PCR reaction with ZNA probes, and by repeating the reactions, we confirmed the reliability of this new approach. It is now recommended to use ZNA probes, as an alternative to MGB probes, to increase the probe Tm value and its binding to target DNA.

Figure 1

Amplification plot data of three samples. The data gathered through the reaction at the end of each PCR cycle. The red and blue lines indicate the base line of T and C alleles respectively. The number above each base line indicates the delta Rn value. A) TT homozygote; B) TC heterozygote; C) CC homozygote.

Figure 2

Final result of a real-time PCR run for 24 samples, which is collected at post-PCR stage. Red, green and blue dots represent TT homozygote, TC heterozygote and CC homozygote samples, respectively.