Design and constructing pBGGT Plasmid: a carrier plasmid for Betathalassaemia gene targeting

Background: Most of molecular biology studies depend on making gene constructs. Although commercial plasmids are widely used for this purpose, sometimes due to the nature of the restriction sites or need of multiple subcloning, usual restriction sites available in original multiple cloning sites (MCS) of the plasmids could not be easily used, if not impossible at all. Here, we report an easy and fast method to construct suitable plasmid with a new MCS for constructing a 16kb gene targeting plasmid.
Methods: Firstly, the suitable MCS was designed by studying the sequence of desired gene fragments in Gene runner software. Two partial complementary 74 base long oligonucleotides were designed and constructed to make this MCS. The original pUC19 MCS was replaced with the new one by enzymatic digestion of the plasmid and removal of the MCS, followed by adding the two complementary oligonucleotides and ligating the construct and transforming it into Ecoli TOP10 F'. The new plasmid was then purified and sequenced by M13 forward and reverse primers.
Findings: Synthesis of two 74 base polynuclotides was successful, and these polynucleotides formed a double stranded fragment which was successfully inserted between HindIII-EcoRI sites of pUC19. Analysis of intermediate step results showed successful progress of cloning reaction. Final analysis of the plasmid by restriction analysis and sequencing the MCS confirmed authenticity of the new plasmid.
Conclusions: The method described here is a fast and easy way to make suitable plasmid out of commercially available plasmids. This process can considerably decrease the time and cost of plasmid construction. Availability of suitable restriction sites in proper order makes it possible to directionally clone the desired gene fragments which is more efficient and excludes screening steps for the right direction of the fragments. The plasmid reported herein is specifically tailored to insert different fragments of a beta-globin gene targeting construct.