Cloning PCR Products Utilizing the T/A Overhang and a Kit

Abstract

The ever expanding identification of new gene family members in recent years has depended in large part on the use of the polymerase chain reaction (PCR) technique. Direct cloning of PCR products into an appropriate vector allows identification of the product by sequencing and characterization of the product’s transcript by Northern hybridization analysis or ribonuclease protection assay. Typically PCR products may be cloned into the vector by cohesive or blunt-end ligation. Cohesive-end ligation traditionally requires the addition of restriction sites to the PCR primers. This necessitates additional enzymatic manipulation and purification steps before the product may be cloned into the vector. Likewise, PCR products amplified with Tag polymerase must undergo further enzymatic manipulation before blunt-end ligation. In addition, blunt-end ligation is a less efficient process than cohesive-end ligation (1).