Is there a recommendation for how long a target sequence for PCR amplification should be?

If there is how do you amplify a target sequence that exceeds the recommended length?


The answer depends a lot on the polymerase you are using. Some are engineered for better processivity, by fusion of a processivity-enhancing domain (Wang et al, Nucleic Acids Res. 2004), but even the best one will struggle after a few tens of kilobases.

You can push the length of the amplicons a little by playing around with conditions but to produce something significantly larger than the average limit of your polymerase, you will usually need to amplify smaller fragments, and then assemble them together. This is the strategy that was used to synthesize the 1079kbp synthetic genome of M. mycoides JCVI-syn1.0, the first "artifical genome" (Hutchison et al, Science 2016); the authors first amplified many fragments of 1.4kbp, and assembled them in vitro. To easily assemble large fragments in vitro, common one-pot enzymatic techniques include the so-called Gibson assembly (Gibson et al, Nature Methods 2009) and Golden gate assembly (Engler et al, PLoS One, 2008) for example.

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