Localizing multiple probes on the same chromosome preparation of Drosophila : A multi - well in situ hybridization technique

In situ hybridization techniques, along with immunocytochemistry allow detection of specific DNA or RNA sequences or proteins in morphologically preserved chromosomes, cells or tissue sections. This technique was originally developed by two independent groups, Pardue and Gall (1969) and John et al. (1969). Since then it has been modified and refined for different applications. Conventional in situ hybridization methods use a single probe on a single chromosome slide. Further, while using enzyme based detections, co-analysis of multiple sequences usually require a number of such slides. However, there can be variations among different chromosome preparations, and it may be difficult to analyze the results together. Here, we describe a “multi-well” technique for in situ hybridization of DNA probes on metaphase and polytene chromosome of Drosophila melanogaser. We have used three genes from Drosophila melanogaster, which have been identified and are being characterized in our laboratory (P28, Acc. No.Z29571; Aminopeptidase P, Acc.No.AJ131920; and Hexokinase, Acc.No.AJ271350) for hybridization on Drosophila chromosome spreads. This method uses polypropylene plastic rings as ‘wells’ around different areas of chromosome spreads, on a single slide, so that multiple probes can be used in different wells at the same time. As shown in Table 1, commercially available or custom made polypropylene rings (which are autoclavable) of different diameters can be used. The metaphase and polytene chromosome spreads were prepared according to standard protocols (Ashburner, 1989). The chromosomal DNA was denatured by incubating the slides for 90s in 0.1N NaOH at room temperature, followed by washing with 2×SSC and subsequent dehydration in increasing alcohol grades. Once air dried, the slides were immediately screened under a microscope to locate a r e a s h a v i n g g o o d chromosome spreads (such areas can also be pre-located and marked). A thin layer of a special adhesive was applied to the lower plastic surface of the wells and using forceps and using a forceps, they were carefully placed on the slide (Figure 1), encircling good spread. The smallest well used (3mm inner diameter) could cover around 25-30 metaphase chromosomes and 5-10 polytene chromosomes, depending on the method and quality of preparation. After about 30 seconds of setting, the slides were pre-warmed to the hybridization temperature and the denatured probes [labeled with either Digoxygenin (Roche, Germany) or the fluorescent nucleotide Cy3 (Amersham, UK)] were added to individual wells Table 1. Dimensions of the wells used and respective hybridization buffer volumes.