Sulfur atom modification on thymine improves the specificity and sensitivity of DNA polymerization and detection

Although accurate base-pairing ensures specificity of molecular recognition, DNA polymerization and DNA amplification, there are many non-specific pairings that arise from mismatched pairs, such as the T/G wobble pair. We have found that by using 2-S-TTP (^STTP), we can minimize T/G mismatch, improve the DNA polymerization specificity and enhance the detection sensitivity (up to 20 fold), without significantly compromising the polymerization efficiency (the extension rate ratio of TTP vs.^STTP is 1.08). With the ^STTP strategy, DNA polymerization is more specific and allows the detection of pathogens (such as COVID-19) in single digits (up to 5 copies), which is not possible with conventional RT-PCR. We have discovered that ^STTP can generally promote much higher specificity and sensitivity in DNA polymerization and nucleic acid detection than canonical TTP.