Rapid Cloning of PCR-Derived RAPD Probes

摘要

The random-amplified polymorphic DNA (RAPD) technique is one of the most useful methods for species identification and studies on the genetic structure of populations of micro- and macro-parasites. This method generally provides numerous markers thatmust be cloned and labeled to be used as probes. 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. One that is not described in the paper by Hengen 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 forthe 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.