Polymerase cycling assembly

While in PCR the customary size of oligonucleotides used is 18 base pairs, in PCA lengths of up to 50 are used to ensure uniqueness and correct hybridization.

As well as the basic requirement of having to be able to tile the entire target sequence, these oligonucleotides must also have the usual properties of similar melting temperatures, hairpin free, and not too GC rich to avoid the same complications as PCR.

It is critical that there is complementarity between all the fragments in some way or a final complete sequence will not be produced as polymerase requires a template to follow.

After this initial construction phase, additional primers encompassing both ends are added to perform a regular PCR reaction, amplifying the target sequence away from all the shorter incomplete fragments.

[3] Ligation independent cloning (LIC) is a new variant of the method for compiling several DNA pieces together and needing only exonuclease enzyme for the reaction.