Comparison of Fluorescence in Situ Hybridization and Primed in Situ Labeling Methods for Detection of Single-Copy Genes in the Fungus Ustilago maydis

Shuxian Li, Charles P. Harris, Sally A. Leong
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引用次数: 10

Abstract

Li, S., Harris, C. P., and Leong, S. A. 1993. Comparison of fluorescence in situ hybridization and primed in situ labeling methods for detection of single-copy genes in the fungus Ustilago maydis. Experimental Mycology 17, 301-308. This report describes the use of fluorescence in situ hybridization for detection of single-copy genes in the fungus, Ustilago maydis . Either biotin- or digoxigenin-labeled DNA probes were hybridized to target nuclei of sporidia and subsequently rendered fluorescent by successive treatments with fluorescein (FlTC)-labeled avidin and biotinylated anti-avidin antibody or with anti-digoxigenin-FlTC or anti-digoxigenin-rhodamine. These results showed that the digoxigenin-based hybridization can be used successfully to specifically detect single-copy genes in nuclei of U. maydis with either 3.6- or 6.7-kb genomic DNA probes. By contrast, the biotin-based hybridization technique gave rise to nonspecific background. Primed in situ labeling with fluorescein-12-dUTP was also used successfully to specifically detect a 1.5-kb fragment of single-copy genomic DNA in nuclei of U. maydis sporidia. This technique has the advantages of lower staining background, shorter time of analysis, and a higher efficiency of hybridization of shorter DNA probes to target DNA, when compared to traditional in situ hybridization.

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荧光原位杂交与引物原位标记法检测麦氏黑穗菌单拷贝基因的比较
李,S.,哈里斯,C. P.,梁,S. A. 1993。荧光原位杂交与引物原位标记法检测麦氏黑穗菌单拷贝基因的比较。真菌学通报,17(3):391 - 398。本报告描述了荧光原位杂交技术用于真菌黑穗病菌单拷贝基因的检测。将生物素或地高辛标记的DNA探针与目标孢子虫细胞核杂交,随后用荧光素(FlTC)标记的亲和素和生物素化的抗亲和素抗体或抗地高辛-FlTC或抗地高辛-罗丹明连续处理,使其呈现荧光。这些结果表明,基于地高辛的杂交技术可以成功地特异检测出马氏菌细胞核中的单拷贝基因,其基因组DNA探针长度分别为3.6 kb和6.7 kb。相比之下,基于生物素的杂交技术产生了非特异性背景。荧光素-12- dutp引物原位标记也成功地特异检测了马氏孢子虫细胞核中1.5 kb的单拷贝基因组DNA片段。与传统的原位杂交相比,该技术具有染色背景低、分析时间短、更短的DNA探针与目标DNA杂交效率高的优点。
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