Rapid cloning of PCR-derived RAPD probes.

The random-amplified polymorphic DNA (RAPD) technique (5,7) is one of the most useful methods for species identification and studies on the genetic structure of populations of microand macro-parasites. This method generally provides numerous markers that must be cloned and labeled to be used as probes (1,4). These probes can be used to test the specificity and the polymorphism of the RAPD markers. The RAPD products can be cloned into plasmid using T vectors that have a thymidine residue at the 3′ end of the linearized form. This T tailing allows the ligation between the vector and the A-tailed polymerase chain reaction (PCR) products. There are several problems with using these T vectors (2). One that is not described in the paper by Hengen (2) is that the use of purification kits to extract the bands from agarose gels considerably reduces the cloning efficiency. Best results were obtained by electroelution of the bands, but this method is tiresome when many bands are to be cloned. This paper describes a method for the rapid cloning of RAPD products with high efficiency. The amplified fragments are first purified from the PCR mixtures with a rapid procedure, cloned together in a T vector and the white colonies screened without preparation of the plasmid. Finally, the plasmids that contained inserts with different sizes are selected. Genomic DNA was extracted with phenol/chloroform (3) from individuals of Haemonchus contortus, a trichostrongyle nematode parasite of small ruminants. Each reaction mixture (50 μL) for RAPD contained 50 ng DNA, 5.0 μL Taq DNA polymerase buffer (Appligene, Illkirch, France) containing 1.5 mM MgCl2, 80 μM each dNTP, 0.5 U Taq DNA polymerase and 0.5 pmol 10-mer primer. The RAPD cycle was carried out for 50 s at 92°C, 30 s at 35°C and 90 s at 72°C for a total of 35 cycles followed by one extensive polymerization reaction (10 min at 72°C). PCR was performed in a thermal cycler (MJ Research, Watertown, MA, USA). For control, 10 μL of PCR products were separated by electrophoresis in a 1.5% agarose gel, and 8–14 bands (0.1–1.7 kb) were generally observed (Figure 1A). No electrophoresis was required for cloning these bands. The RAPD reaction mixture was directly purified from primers, nucleotide, polymerase and salts using the QIAquick PCR Purification Kit (Qiagen, Chatsworth, CA, USA) according to the manufacturer’s instructions. The fragments in the mixture were then cloned in the pGEM-T Vector (Promega, Madison, WI, USA). The optimal insert/vector ratio that gave good ligation was 10–80 ng purified PCR products plus 50 ng vector. Ligation and transformation of ligated products in JM109 High Efficiency Competent Cells (Promega) were done according to the supplier’s instructions. The ratio of blue/white colonies was