Simplified Method for Ligase-Free Cloning of PCR Products

Cloning of polymerase chain reaction (PCR) products into plasmid vectors by conventional methods, relying either on recognition sequences built into the primers or taking advantage of the template-independent terminal transferase activity of Taq DNA polymerase to add an extra adenine to the 3′ end of the product, are often difficult (1,10,12). In recent years, numerous cloning strategies (10,13) have been developed to overcome these problems. We were using one such technique, the ligase-free subcloning method (13), to clone PCR products into plasmid vectors. However, we found that the method is still time-consuming and, above all, leads to a considerable number of wrong products. On the average, 30% of the recombinant plasmid products were found to be wrong. These were probably formed mostly during the long heterologous reannealing step of the method. Avoiding this step and transforming cells directly with a single second PCR product were attempted (13), but the structure of the few plasmids obtained in this way was not examined. There are many reports in the literature of transformations with linear plasmid DNA (3,11,14). Depending on both the DNA and the type of host cell used, a low yield of transformants and frequent modifications of the transformed DNA by exonucleases or recombination events, were observed after in vivo ligation (14). We have applied a simplified ligasefree cloning method, which requires only three primers and rests essentially on a direct transformation of E. coli cells with linear DNA produced in a second PCR. Plasmids containing cDNA fragments from glyceraldehyde3-phosphate dehydrogenase (GAPDH) and liver, muscle and heart isozymes of rat 6-phosphofructo-2-kinase/fructose2,6-bisphosphatase (PFK-2/F2,6Pase) (Figure 1) were constructed. Sequences of the primers used are given in Table 1. Primer b was used for the synthesis of first-strand cDNA from total RNA by reverse transcription (Figure 1). Primers a and b were used for the first PCR. Primer b contains, in addition to specific sequences of about 25 nucleotides at its 3′ ends, additional 5′end sequences of 24 nucleotides, which are identical to the 3′ end of linearized pT3T7BM (digested with HincII). In the second PCR, the initial amplified fragment and linearized pT3T7BM were annealed and amplified using primers a and c (Figure 1). Primer c, 5′TTTAGTGAGGGTTAATTTCGAG-3′, a downstream primer and identical in all cloning reactions, is complementary to the (+)-strand of the plasmid. The linear plasmid produced was directly used to transform competent E. coli TG2 cells (7). Typically, about 250