PCR-based WGA

There are 3 main PCR-based WGA techniques. These are degenerate oligonucleotide PCR (DOP-PCR) (1), primer extension preamplification (PEP) (2) or derivatives thereof, and adaptor-ligation PCR (3). The main difference between the techniques is that PEP uses a preamplification step to add primer binding sites to small DNA fragments for later WGA by PCR, while adaptor-ligation PCR uses adaptors ligated to small DNA fragments to create PCR primer binding sites. PEP utilizes random primers and a low PCR annealing temperature. Less frequently used today, DOP-PCR uses semi-degenerate oligonucleotides (i.e., CGACTCGAGNNNNNNATGTGG) and an increasing annealing temperature. The use of Taq DNA polymerase in both techniques limits the fragment lengths to 3 kb (average fragment sizes are 400–500 bp) and also introduces a number of errors into the sequence. Furthermore, these techniques have been found to exhibit incomplete genome coverage and amplification bias – where a sequence is overrepresented in the amplified DNA due to preferential binding of the primers to specific regions.