Indian Journal of Experimental Biology Vol. 37, October 1999, pp. 1046-1048 A non-radioactive method for mapping restriction fragment length polymorphic genetic markers in Anopheles gambiae N Pradeep Kumar Vector Control Research Centre (ICMR), Pondicherry 605 006, India Received 13 May 1999; revised 14 July 1999 A non-radioactive method for in situ hybridisation of Restriction Fragment Length Polymorphic (RFLP) markers to the polytene chromosome of Anopheles gambiae, the important malaria vector, which yielded good readable quality of chromosomal bands is reported. The methodology adopted was a Biotin-Streptavidin-Alkaline Phosphatase system which yielded fluorescent signals when stained with dyes such as Nitro Blue Tetrazolium and Bromo Chloro Indolyl Phosphate. Anophelines are the vectors of malaria, the major tropical disease afflicting between 300 to 500 million population globally'. Currently, molecular biological tools are being employed to analyze the genome of these vector species with an ultimate objective of devising an effective control strategy of this vector borne disease 2 • The construction of the genome map of the most important vector of malaria viz., Anopheles gambiae has already been accomplished using genetic markers such as microsatellites 3 and is being undertaken using Random Amplified Polymorphic DNAs (RAPD)4 and Restriction Fragment Length Polymorphisms (RFLPi. Anopheline mosquitoes have excellent polytene chromosomes, both in the germinal and somatic cells, thus offering material for physically mapping the genome. Physical mapping of DNA markers to polytene chromosomes haS been reported in Drosophila melanogaster using radioactive and non-radioactive methodologies 6 . 7 • Also, there exist reports on attempts in Ali. gambiae 8 and An. albimanus 9 to physically map genetic markers to polytene chromosomes. However, readability of the chromosomal bands after hyblidisations had been difficult owing to loss' of clarity during the process. The present report is on the standardisation of a non-radioactive methodology for mapping a group of identified RFLP markers 5 in An. gambiae to the polytene chromosomes of the species. The readability of the banding pattern of the chromosomes was given much importance. The RFLP markers isolated by screening the genome of different strains of An. gambiae, using cDNA library clones of species 5 were used in the study. The markers varied from 2.0-11.4 Kilo basepairs (Kb) of An. gambiae DNA cloned into 'lambda' vectors. These were subcloned into plasmid vectors (pUC 19) to an optimum size 8 of 2.0-4.0 Kb, nick translated and labelled with biotin-14-dA TP (Bionick DNA labelling, GmCO, BRL). The labelled probes were dissolved in 800 microlitre of 2X hybridisation buffer and purified through Sephadex G-50 columns. The chromosomes of 'SUOKOKO' strains of All. gambiae, reported to have iso-sequential ovarian nurse cell polytene chromosomes 10 were prepared from semi- gravid specimens reared in the laboratory. The mosquito specimens were dissected in a drop of cold Carnoy's fixative. The ovaries were transferred to a drop of 50% propionic acid for 10 min and then to a fresh slide treated with 3-aminopropyl triethoxysilane. A siliconized cover glass was placed over it and was squashed between a folded filter paper. After incubation of the slides over dry ice for about 10 min, coverslip was flipped off using a sharp razor. This was immediately immersed in cold 50% ethanol for 5 min, hydrated in Phosphate Buffered Saline (PBS). The chromosomes were de-proteinized by immersing the slides in protienase K dissolved in PBS (40 Jlg/ml), at 37°C for 2 min and were treated with 4% paraformaldehyde solution In PBS at room temperature. 35 microlitre of the probe (concentration of approx., 0.5 Jlg DNA/ml) was mixed with equal amount of 20% dextran sulfate solution. The mixture was added to the slide with the chromosomal preparation and was covered with a cover glass. The slides were kept on a slide wrumer maintained at 100°C for denaturation of chromosomal and probe DNA for 10 min. The edges of