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How to design primers for PCR

Primers are short, single-stranded nucleic acids that initiate DNA synthesis. During PCR reactions, they anneal to the plus and minus strands of the template DNA, flanking the sequence that needs to be amplified. To design primers, you usually use a primer design tool. The tool helps you customize your primers based on specific parameters such as primer length, melting temperature or GC content. This video will explain what the optimal values for each of these parameters are, and how they affect your PCR assay. For more information: https://www.integra-biosciences.com/e... Primer length The optimal primer length is between 18 and 24 base pairs. Shorter primers may lead to non-specific amplification, while longer primers anneal less efficiently to the template DNA, resulting in a lower amount of PCR product. Target sequence The target sequence to be amplified should ideally be between 100 and 3000 base pairs for standard PCR assays, and 75 and 150 base pairs for qPCR assays. Longer sequences usually need special enzymes and reaction conditions to ensure that they are completely and specifically amplified. Melting temperature (Tm) The primer melting temperature is the temperature at which half of the primers dissociate from the template DNA. It's typically between 50 and 60 degrees Celsius. Annealing temperature (Ta) The primer annealing temperature is the temperature needed for the specific annealing of the primers during the PCR reaction. The theoretical annealing temperature can be calculated using the lower melting temperature of the primer pair and the melting temperature of the PCR product. To find the optimal annealing temperature, perform a gradient PCR starting a few degrees below the calculated annealing temperature and ending a few degrees above. After amplification, run a gel. The sample resulting in the clearest band contains the largest quantity of PCR product, making its annealing temperature the optimal one for your primers. GC content GC bonds are harder to break than AT bonds because GC base pairs are linked by three hydrogen bonds and AT base pairs by two. This means that a higher GC content ensures a more stable binding between the primers and the template DNA. The optimal GC content of a primer lies between 40 and 60 %. Repeats Avoid runs of four or more single bases or dinucleotide repeats as they can cause mispriming. Cross homology If a primer is homologous to a template DNA sequence outside the region of interest, these sequences will be amplified too. Therefore, you should always test the specificity of your primer design against genetic databases. Secondary structures There are three different types of secondary structures, also called primer dimers, that can form during a PCR assay: hairpins, self-dimers and cross-dimers. Your PCR product yield will be less if secondary structures form and remain stable above the annealing temperature of your reaction. This is why your primer design tool should be able to check for, and warn you of stable secondary structures. Mismatches and degenerate primers Mismatches are primer bases that aren't complementary to the target sequence. If mismatches negatively impact the performance of your PCR, degenerate primers can help. Degenerate primers have several different nucleotides in some of their positions. For example, instead of A you could have an equal concentration of A and T in a certain position. Video content [00:00] – What are PCR primers [00:22] – How to design primers [00:41] – Primer length [01:00] – Target sequence [01:22] – Melting temperature (Tm) [01:47] – Annealing temperature (Ta) [02:43] – GC content [03:06] – Repeats [03:16] – Cross homology [03:33] – Secondary structures [04:26] – Mismatches and degenerate primers [05:09] – More information #INTEGRA #INTEGRABiosciences #pcr #primerdesign