IMMUNOLOGICAL METHOD FOR MAPPING GENES ON DROSOPHILA POLYTENE CHROMOSOMES
IMMUNOLOGICAL METHOD FOR MAPPING GENES ON DROSOPHILA POLYTENE CHROMOSOMES
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DOI：
10.1073/pnas.79.14.4381
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发表时间：
1982-01-01
期刊：
影响因子：
--
通讯作者：
WARD, DC
中科院分区：
文献类型：
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作者：
LANGERSAFER, PR;LEVINE, M;WARD, DC
描述了一种用于定位与果蝇多线染色体原位杂交的DNA序列的方法。该方法利用生物素标记的TTP类似物，其可以通过切口平移酶促掺入DNA探针中。原位杂交后，探针中的生物素分子作为抗原，与亲和纯化的兔抗人球蛋白抗体结合。然后，通过荧光标记的山羊抗兔IgG进行荧光分析，或通过与辣根过氧化物酶结合的抗兔IgG抗体进行细胞化学分析，检测杂交位点。当与Giemsa染色结合时，免疫过氧化物酶检测方法提供了适合详细细胞遗传学分析的永久记录。与传统的放射自显影方法相比，这种免疫学方法具有4个优点：确定杂交位点所需的时间明显缩短;生物素标记探针化学稳定，可重复检测数月;生物素标记探针产生的背景噪声比放射性标记探针小;并且分辨能力等于并且通常大于放射自显影所获得的分辨能力。
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-labled goat anti-rabbit IgG, or cytochemically, by using an anti-rabit 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 4 advantages over conventional autoradiographic procedures for detecting in situ hybrids: the time required to determine the site of hybridization is decreased markedly; biotin-labeled probes are chemically stable and give reproducible results for many months; biotin-labeled probes appear to produce less background noise than do radiolabeled probes; and the resolving power is equal to and often greater than that achieved autoradiographically.