Amplification and stability of recombinant plasmid molecules with a very large insert of foreign genetic material

The capacity of bacterial plasmid vector pAT 153 for harbouring and for amplifying in a stable way a very large insert of foreign genetic material was screened. The DNA of a tree shrew herpesvirus (strain 2) with a molecular weight of about 130 megadalton (200 kilobasepairs) consisting of a long unique DNA sequence without direct and inverted repeats (greater than 40 basepairs), was used for molecular cloning. A successful insertion and stable amplification of a DNA fragment with 49 megadalton (74 kilobasepairs) cloned in Eco RI site of pAT 153 was performed, which resulted in the construction of recombinant DNA plasmid pTH2-E-C1. The stability of the recombinant plasmid pTH2-E-C1 with a super insert of 74 kilobasepairs (49 MD) indicates that the bacterial plasmid vector pAT 153 has really a high capacity for harbouring foreign genetic material.