Short sequences can be introduced into a target gene by adding the new DNA sequence to the 5'-end of the primer or primers used for the PCR reaction.
If the insertion is to be added to the end of the target gene, a simple PCR amplification with the extended primer will achieve this outcome.
If the insertion is to be placed at a specific location within the target gene, overlap extension-splicing can be employed. By using overlapping primers A and B, that contain the insertion sequence at their 5'-ends, for the primary PCR amplification it is possible to insert any short new sequence anywhere within the target gene. However, the length of oligonucleotides that can be synthesized using current chemistry limits the insertion size. It is technically difficult to synthesize oligonucleotides longer than 80-100 nucleotides. Since the 3'-segment (approximately 20 nt) of the oligonucleotide must act as an efficient PCR primer, the insertion sequence could be 80-100 nucleotides.
By using a high-fidelity proofreading DNA polymerase and a limited number (10 cycles) of amplification cycles per insertion, it is not necessary to clone and sequence the product after every round of insertion mutagenesis.
Horton, RM, Hunt HD, Ho, SN, Pullen, JK, Pease, LR (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77: 61-68.