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The most complex step in multiplex design is the selection of primers. Primer-primer interaction can result in decreased yield and artifacts. Computer programs are used to design primers and determine primer interaction. A set of primers must perform under a single amplification environment. It is important that the primers selected for the multiplex are designed based upon melting temperatures and locus specificity.
Read more about Locus Selection from this course.
To achieve balance (product yield) across all loci, the quantity of an individual primer can be adjusted within the multiplex set. This part of the process is highly empirical and can be very time consuming.
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