Designing primers for PCR amplification
Designing primers is the first stage of many genetical and biological experiments. Primers are short oligonucleotide sequences that bind to the genomic sequence under PCR conditions. Well designed allow identification of genetic mutations or genomic fragments. The template, i.e. the identifiable sequence, can be any organism whose reference sequence is known and described in databases. Primers are commonly used in human genetic diagnostics, but they are equally often used to identify differences between species of different organisms.
Designing starters is not an easy art. To obtain the desired product in the PCR reaction, primers must have certain characteristics and undergo a number of verifications. The first most important feature is specificity. A specific primer is one that does not bind anywhere else in the genome except for the target sequence. Designing primers can be problematic when we encounter a pseudogene sequence characteristic of many genes in the human genome. A pseudogene is a gene whose sequence is identical to its functional copy, i.e. one that encodes a protein. Pseudogenes, apart from their presence, have no function, or at least their function is not fully understood. Some genes can have from few to dozen pseudogenes. Pseudogenes amplified in the PCR reaction interfere with its result.
Starters must also have an appropriate melting point temperature - preferably 60ºC, and a length of 17-28 bp. They also cannot have affinity with each other and create primer dimers. However, this disadvantage can be easily eliminated by testing various PCR reaction conditions. Finally, primers should not contain popular genetic variants in their sequence. The presence of such variants may prevent the primer from attaching to the patient's DNA and thus may not give the desired product.
Going through all the stages described above guarantees well-designed primers. In our offer you will find a service for designing primers for PCR reactions and PCR multiplex. We also invite you to check for the degenerated (universal) primer design service.
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