Cloning of DNA repetitive sequences from the genome of Culex quinquefasciatus for the development of DNA probes.

Abstract

Filariasis is a major public health problem confined mainly to tropical and sub tropical countries, around the world (Walls and Schantz, 1986). Estimates show that a sizable proportion of the world’s population is affected with disease (UNDP/WHO, 1986). Filariasis is transmitted to human by the infective bites of mosquito vectors. The identification of individual species, a requirement of epidemiologic studies and control programs, has traditionally relied upon techniques such as mating incompatibility, isozyme analysis, cytological methods, hydrocarbon analysis, etc.. Owing to the limitations of these techniques, the last few years have seen many developments in DNA based technologies for identification. These methods ultimately rely upon either the use of DNA probes in hybridization assays or the polymerase chain reaction (PCR). This study reports the cloning and isolation of recombinant clones containing repetitive sequences of C. quinquefasciatus for the development of specific DNA probes which could be used for the identification of mosquito adult, larvae and pupae. Culex quinquefasciatus genomic DNA was partially cleaved with the restriction enzyme Sau 3A-1 to obtain the maximum number of fragments in the size range 0 - 9 kb. These fragments were cloned into the bacteriophage vector ƛZap and packed in vitro. The library was then amplified and percentage of recombinants were determined. To isolate recombinant clones containing repetitive sequences of C. quinquefasciatus for the development of DNA probes, an aliquot of the library was plated out and plaque lifts obtained were screened with P-labeled C. quinquefasciatus genomic DNA. Plaques giving strong hybridization signals on filters were marked and picked into to SM buffer. These repetitive recombinant clones were stored at 4°C for further analysis.