Bergey D R, Stelly D M, Price H J, McKnight T D
Department of Biology, Texas A & M University, College Station.
Stain Technol. 1989 Jan;64(1):25-37. doi: 10.3109/10520298909108040.
A modified procedure for in situ hybridization of biotinylated probes to meiotic chromosomes of cotton has been developed with high retention of squashed cells on slides, preservation of acid-fixed chromosome morphology, exceptionally low levels of background precipitate at nonspecific hybridization sites and improved photomicrographic recording. Salient features of the techniques include pretreatment of slides before squashing, cold storage of squash preparations, and use of interference filters for distinguishing precipitate from chromatin. A cloned 18S/28S ribosomal DNA fragment from soybean was biotinylated via nick-translation and hybridized to microsporocyte meiotic chromosomes of cotton (Gossypium hirsutum L. and G. hirsutum L. X G. barbadense L.). Enzymatically formed precipitate from streptavidin-bound peroxidase marked the in situ hybridization. In situ hybridization of biotinylated probes to cotton meiotic chromosomes adds the specificity and resoltion of in situ hybridization to the chromosomal and genomic perspectives provided by meiotic cytogenetic analyses. Molecular cytogenetic analyses of meiotic cells offer certain inherent analytical advantages over analyses of somatic cells, e.g., in terms of mapping, and for studying fundamental biological and genetic problems, particularly for organisms that are not amenable to somatic karyotypic analysis.
已开发出一种将生物素化探针原位杂交到棉花减数分裂染色体上的改良方法，该方法能使压片细胞在载玻片上高度保留，保持酸固定染色体形态，非特异性杂交位点的背景沉淀水平极低，并改善显微照相记录。该技术的显著特点包括压片前对载玻片进行预处理、压片制剂的冷藏以及使用干涉滤光片区分沉淀和染色质。通过缺口平移法将来自大豆的克隆18S/28S核糖体DNA片段生物素化，并与棉花（陆地棉和陆地棉×海岛棉）的小孢子母细胞减数分裂染色体杂交。链霉亲和素结合的过氧化物酶酶促形成的沉淀标记原位杂交。生物素化探针与棉花减数分裂染色体的原位杂交，将原位杂交的特异性和分辨率加入到减数分裂细胞遗传学分析所提供的染色体和基因组视角中。减数分裂细胞的分子细胞遗传学分析相对于体细胞分析具有某些固有的分析优势，例如在作图方面，以及用于研究基本的生物学和遗传学问题，特别是对于不适于进行体细胞核型分析的生物体。
