Comparative study of three methods for cloning PCR products

The direct sequencing of the products of polymerase chain reactions (PCR) still presents difficulties and often requires special manipulations, such as the generation of excess single-stranded DNA using asymmetric PCR. Several alternative methods involve cloning PCR products into vector DNA suitable for sequencing analysis. Three of these methods have been compared in the present study. The two direct cloning methods, TA/cloning and the PCR-script system, initially gave large numbers of false positives (60% and 55%, respectively) but the number of false positives was reduced (to 35% and 31%, respectively) by modifying the protocols used. However, ligation of the termini of the digested PCR product in the corresponding digested vector was the most efficient and consequently the most reliable method for routine cloning.