Minimum GC-Rich Sequences for Overlap Extension PCR and Primer Annealing

PCR is a common method to produce desired DNA fragments from templates. The oligonucleotide primers used for PCR must contain annealing sequences that are usually 20–30 nucleotides long and identical to a part of template DNA. However, primers often contain additional sequences at their 5′ ends, which are restriction enzyme sites, recombination targeting sequences, or overlap sequences for fusion PCR. When these additional sequences are attached to their annealing sequences, the annealing sequences can be shortened. Here, we describe universal GC-rich annealing sequences useful for overlap extension PCR and simple in-frame addition of desired sequences.