Molecular cloning is a fundamental method in molecular biology, it means isolation of DNA sequence from any species and insert into a vector propagation.
Cloning is used to generate many numbers of identical copies which helps to analyse gene sequence and its expression. Since its high demand in research many techniques were developed like Gibson cloning (reference) Uni-vector plasmid-fusion system, Golden Gate system etc. In this protocol we worked with two cloning system one is transformation-associated recombination system TAR and second one circular polymerase extension cloning CPEC.In circular polymerase extension cloning (CPEC), double stranded vector and insert are overlapped and extend through polymerase extension mechanism. In CPEC mechanism, both ends of insert and vector shares overhanging sequence. In PCR cycle double stand DNA are denatured and later align during annealing step. The hybridized insert and vector are extended using each other as template to form a complete double stranded plasmid. So a complete double stranded plasmid is formed using only polymerase as enzyme in single tube reaction without restriction digestion, ligation reaction, and single-stranded homologous recombination.
In transformation-associated recombination system (TAR) allows to isolate large DNA fragments from a complex genome to simplify the chromosomal mapping. This mechanism, yeast artificial chromosome (YAC) are constructed by homologous recombination and modified to transfer into E.coli cells. To form YAC, Excess of TAR cloning vector are mixed with genomic DNA fragments. TAR cloning Vector fragments carried two targeting sequence known as hooks which is similar to gene of interest, so when there is any homology between vector and genomic fragment then the recombination will occurred and circular YAC formed.E.coli cells is good choice for its recombinant proteins production and short culturing time.This protocol we performed in two different way (TAR and CPEC cloning) to analyse the protein expression with lacZ gene.