In a qPCR assay, both primers and probes play important roles in amplifying and detecting the target DNA. Primers are short DNA sequences that bind to the specific regions flanking the target DNA sequence, while the probe is a labeled DNA molecule that binds to a specific region within the target DNA sequence. To connect the primer and probe sequences, you would typically design your primers to be complementary to the regions adjacent to the target sequence, and the probe to be complementary to a region within the target sequence. The primers and the probe should not directly connect to each other.

Using a string of T bases (e.g., TTTTT) between the primer and probe sequences is not a common practice in qPCR design. This is because the presence of extra bases between the primer and probe can affect the efficiency and specificity of the PCR reaction.

Instead, the primer and probe sequences should be designed in a way that allows them to work independently and efficiently. The primers should bind specifically to their target regions, and the probe should bind specifically within the target sequence. It is recommended to use specialized software or online tools for primer and probe design, such as Primer3, NCBI Primer-BLAST, or IDT PrimerQuest. These tools consider factors like primer specificity, melting temperature (Tm), GC content, and secondary structure to design primers and probes that work well together and produce reliable qPCR results. By designing the primers and probe appropriately, you can ensure efficient amplification and detection of your target DNA in the qPCR assay.

You do not need to connect the probe and primers. They are designed to bind independently.