Hetero-stagger cloning: efficient and rapid cloning of PCR products.

A variety of methods have been developed for cloning PCR products, including blunt-end cloning (1), restriction cut back (2), ligation-independent cloning (3), uracil DNA–glycosylase (UDG) treatment of uracil-containing deoxyoligonucleotide primers (4,5) and TA cloning (6–8). Blunt-end cloning of PCR products often requires treatment of PCR products to polish the ends (9). Even with treatments, blunt end cloning is inefficient. The key element in improving cloning efficiencies has been the creation of cohesive compatible ends. Other than blunt end cloning, various current available PCR cloning methods differ in how the cohesive ends are created. In the restriction cut back strategy, restriction sites are incorporated in the PCR primers and the cohesive ends are created by restriction digestion after PCR. This procedure involves two drawbacks: (i) extra bases for restriction sites increases the cost and leads to primer dimer formation; and (ii) restriction enzymes function poorly at the ends of the molecules (2). In both the ligation-independent cloning and the UDG cloning strategy, a 12 base overhang is created by treatment of the PCR products with T4 DNA polymerase or uracil DNA–glycosylase respectively. These procedures are highly efficient, but the main disadvantages are complications involved in vector preparation and the added expense of 24 extra bases to the PCR primer set (11). The TA cloning strategy utilizes the terminal extendase activity of Taq DNA polymerase (9) adding an extra base to the 3′ of the PCR products, which is template-independent, but is sequence-specific. A single G is added if the 3′ nucleotide on the fragment is a G, but an A is added if the 3′ nucleotide is a C, T or A, with very low efficiency of additions when the 3′ nucleotide is an A (10). As a result, TA cloning can only clone molecules with extended A. Most importantly, TA cloning is not useful with the newer proof-reading DNA polymerases for PCR amplifications such as Pfu or Pwo thermostable DNA polymerases (11). In this communication, I present a novel method for PCR cloning termed ‘hetero-stagger PCR cloning’ (Fig. 1). The procedure is based on the fact that related PCR products of different lengths can be obtained by preparing related primers of different lengths. These related PCR products are mixed, heat-denatured, and annealed to form heteroduplex thereby molecules with cohesive ends can be generated. These molecules with cohesive ends can be readily cloned into compatible vectors (Fig. 1). PCR products amplified by using Taq polymerase as well as by using proof-reading thermostable enzymes can be cloned by this procedure. Three extra bases, GGG, are added to half of the customized PCR primers at the very 5′ ends. The other half of the primers is made as determined by the target sequences without extra GGG. Two PCR reactions are set up: one PCR reaction with primer pair of GGG-upper primer plus lower primer, and the other PCR reaction with upper primer plus GGG-lower primer. The PCR products of the two reactions are identical except that each one harbors three extra Figure 1. The hetero-stagger cloning strategy. Two PCR reactions were set up: one with GGG-upper primer (GGG + arrow) and lower primer (arrow), and the other with upper primer (arrow) and GGG-lower primer (GGG + arrow). The two PCR products are different only at the ends by 3 bp as indicated. These two PCR products were mixed, heat-denatured, and re-annealed. Four annealing products were expected, each at a 25% probability. Two of the four annealing products are molecules with cohesive ends: one with GGG 5′ overhang and the other with CCC 3′ overhang. These molecules with cohesive ends can be readily cloned into compatible vectors.