Immunological method for mapping genes on Drosophila polytene chromosomes.

TLDR

The study presents a method for localizing DNA sequences hybridized in situ on Drosophila polytene chromosomes. The technique incorporates a biotin‑labeled TTP analog into probes by nick‑translation, hybridizes them to chromosomes, then uses antibiotin antibodies and either fluorescein‑labeled or peroxidase‑conjugated anti‑rabbit IgG for fluorimetric or cytochemical detection, and Giemsa staining yields a permanent cytogenetic record. The immunological approach reduces detection time, yields chemically stable, low‑background, reproducible probes, and provides resolving power equal to or better than autoradiography.

Abstract

A method is described for localizing DNA sequences hybridized in situ to Drosophila polytene chromosomes. This procedure utilizes a biotin-labeled analog of TTP that can be incorporated enzymatically into DNA probes by nick-translation. After hybridization in situ, the biotin molecules in the probe serve as antigens which bind affinity-purified rabbit antibiotin antibodies. The site of hybridization is then detected either fluorimetrically, by using fluorescein-labeled goat anti-rabbit IgG, or cytochemically, by using an anti-rabbit IgG antibody conjugated to horseradish peroxidase. When combined with Giemsa staining, the immunoperoxidase detection method provides a permanent record that is suitable for detailed cytogenetic analysis. This immunological approach offers four advantages over conventional autoradiographic procedures for detecting in situ hybrids: (i) the time required to determine the site of hybridization is decreased markedly, (ii) biotin-labeled probes are chemically stable and give reproducible results for many months; (iii) biotin-labeled probes appear to produce less background noise than do radiolabeled probes; and (iv) the resolving power is equal to and often greater than that achieved autoradiographically.