If all targets are still covered, primers are distributed to the defined number of pools. Primers that do not overlap variable positions with defined parameters (the frequency of the alternative allele and the distance to the 3'-end) are saved to the new XLS-file (“draft” file 3), others are removed. The ASPE method involves two phases, an enzymatic reaction that determines the target genotype followed by a capture on solid microsphere surface for detection. If all target regions are still covered, the primers are verified for overlapping genome regions with variable positions. Allele Specific Primer Extension (ASPE) is a solution based, sequence specific enzymatic reaction technology that can be used to assay multiple SNPs in a single tube. ![]() Primers that do not hybridize to many targets and do not form non-target amplicons are saved to distinct XLS-file (“draft” file 2), others are removed. bioinformatic resources such as PrimerPlex are able to assist with the process of multiplex PCR design. If all target regions are covered, the primers are verified for non-target hybridization and amplification. of the primers require careful optimisation. Finally, before ordering or synthesising the. If it is necessary, all primers are verified for the forming secondary structures due to the additional adapter sequences at the 5'-ends of primers. Bioinformatic resources, such as primerplex, are also able to assist with the process of multiplex PCR design. Multiplex assays facilitate amplification of multiple targets in a. ![]() NGS-PrimerPlex tries to design primers for all input regions saving primers constructed to the XLS-file (“draft” file 1). PrimerPlex is an efficient and sophisticated tool for designing oligos for multiplex assays. regions of a reference genome), however, such region coordinates can be automatically obtained with getGeneRegions.py script. The primer design process starts from the list of sequence regions (e.g. NGS-PrimerPlex supports distribution of all primers into any number of multiplexes (unlike e.g. Solid arrows denote obligatory steps dotted arrows denote optional steps that can be applied by the user.
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