Advances In The Methods Of PCR-based Mutagenesis With Central Overlap Primers (COP)

With the development of PCR-based mutagenesis in vitro,the number of primer varied from at least 4 primers of Recombination PCR to 3 primers of Megaprimer PCR.When it comes to QuikChange Mutagenesis PCR,there are only 2 primers in use.It is improved from 3 rounds of PCR to only one round in Quikchange PCR and Inverse PCR.The great advances in PCR technology have saved a lot of time and energy for scientists.Recently,it is reported that DNA fragments of insertion,substitution and deletion mutation are generated according to methods based on QCM-PCR.However, it is difficult to yield positive clones because of low output efficiency.On the other hand,the Inverse PCR combined with a tail-to-tail primer design requires T4 DNA ligase enzyme to ligate PCR products,which makes the positive rate relatively low.We aim to find out an easy and effective method to perform gene deletion, insertion and substitution mutation.On the base of numerous research and experiments,a new mutagenesis method is initiated,which uses a pair of central overlap primers(COP) ground on the principle of Inverse PCR.The method is just like the QuikChange site-directed mutagenesis method except for its special primer. This pair of primers is composed of a long primer and a short primer which is central overlapped.Both of the Tm of 5'sequence in long primer and Tm of 3'sequence in short primer are above 66℃.The Tm of short primer is almost half of that of long primer.It is convenient to perform insertion or substitution mutation of DNA fragments larger than 20bp,In addition,it is highly applicable in research about the deletion mutation of DNA fragments larger than 2000bp.This method of primer design can be used to construct mutant plasmids with great ease and speed.It applies to various gene cloning techniques,such as multiple-point mutation,deletion,replacement,insertion,generating positive rates of higher than 85%.