Primers & Probes

    In molecular diagnostic technology, primers and probes are the core. Primers are artificially synthesized oligonucleotide sequences. As the key guiding molecules for the initiation of nucleotide polymerization, their length is generally between 18 and 27 base pairs, which is related to the Tm value and specificity. If they are too long (greater than 38 base pairs), the extension temperature will exceed 74°C, which is beyond the suitable reaction temperature range of Taq DNA polymerase, affecting enzyme activity and DNA replication. 

    In the PCR process, primers are crucial and provide a starting point for DNA polymerase to initiate replication. One primer is complementary to the template chain at one end of the target region, and the other is complementary to the other end, indicating the starting position for the polymerase. After PCR is initiated and the temperature rises to unwind the DNA template chain into a single strand, the primer binds to the specific region according to the principle of base complementary pairing. The DNA polymerase recognizes the binding site, adds nucleotides from the 3' end of the primer, and extends the new chain from the 5' to the 3' end. The cycle is repeated to achieve exponential amplification of DNA fragments. Primers are like the cornerstone of building a high-rise building, ensuring the efficient advancement of PCR. 

    The probe is a labeled nucleic acid sequence that specifically binds to the target nucleic acid sequence through molecular hybridization to accurately detect the target. It is a key "weapon" for molecular diagnosis and can be a DNA or RNA sequence with a marker like a "signal light". There are many nucleic acid molecules in biological samples. When there is no label, the weak signal of the probe binding to the target nucleic acid is easily submerged. With the marker, the probe binds to the target sequence and can emit an easily captured signal through radioactive isotope autoradiography, fluorescent dye luminescence, or chemical color development, helping researchers understand the target nucleic acid.