The denaturation stage of PCR takes about 1?2 minutes. The rmocycler n lowers temperature to about 50° to 60°C, which allows short, oligonucleotide primers to anneal to ir complementary sequences on single-stranded DNA molecules. The annealing stage of PCR lasts about 30 seconds. Hence, whole cycle of gene amplification requires 30 minutes.

Recognition site is sequence of DNA where DNA is cleaved. The presence of more than one recognition site will lead to several cuts within DNA leading to fragmentation of DNA. This will complicate process of gene cloning.