FAQ: How can I design probes for LDR or LCR to maximize specificity?

Probes for LDR (Ligase Detection Reaction) and LCR (Ligase Chain Reaction) should be designed so that all anneal within a narrow temperature range (ideally < 2°C), and should have annealing regions 15-30 bases long. Typically, the best reaction temperature for highest fidelity will be approximately between 2°C below and 2°C above the Tm for a given probe set, calculated according to the buffer conditions used. Use of the Thermostable Ligase Reaction Temp Calculator is highly recommended for selection of an incubation temperature. However, the optimal reaction temperature for a given probe set must be determined empirically.

When using HiFi Taq DNA Ligase, the SNP to be discriminated can pair with either the 3´ base of the upstream probe (providing the 3′-hydroxyl to the ligation junction) or the 5′ base of the downstream probe (providing the 5´-phosphate to the ligation junction), as HiFi Taq DNA Ligase exhibits increased discrimination between correct and mismatched base pairs at either side of the ligation junction. Please see the accompanying product information for a detailed analysis of the fidelity of mismatch ligation by HiFi Taq DNA Ligase that illustrates its higher fidelity at both the 3´ and 5´ side of the ligation junction and specific base pair discrimination ratios.

For more information regarding ligase specificity and high fidelity ligases, visit “Substrate specificity and mismatch discrimination in DNA ligases”.