Optimization of TaqMan Probe Design for Short Template Strands

초록

TaqMan probes are essential tools in nucleic acid diagnostics, enabling sequence-specific detection and are widely utilized in liquid biopsies. However, detecting ultra-short cell-free DNA (cfDNA), typically 40-80 bp in length, presents significant challenges for TaqMan probe design. Designing primers for short template strands further restricts the available TaqMan probe length, making it difficult to adhere to the conventional guideline that requires the TaqMan probe's Tm to be 8-10 degrees C higher than the primer Tm. To address these challenges, DNA modifications such as locked nucleic acids (LNAs) and minor groove binders (MGBs) are often employed. However, these approaches increase costs and, in the case of LNAs, can reduce fluorescence signal efficiency due to cleavage inhibition. This study aimed to establish refined guidelines for designing TaqMan probes for short template strands without relying on DNA modifications. We found that maintaining a minimum gap of 2 nucleotides between the primer and the TaqMan probe prevents Taq polymerase cleavage inhibition, thereby ensuring stable amplification signals. Additionally, TaqMan probes with Tm values up to 2 degrees C lower than the primer Tm successfully generated amplification signals that were clearly distinguishable from background signal. These findings provide practical and cost-effective guidelines for designing TaqMan probes for short template strands without relying on DNA modifications. Moreover, these guidelines are expected to extend their applicability beyond short template strands to diverse scenarios in TaqMan probe design.

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