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荧光原位杂交技术使用寡核苷酸探针。

Fluorescent In Situ Hybridization Using Oligonucleotide-Based Probes.

机构信息

Department of Plant Biology, Michigan State University, East Lansing, MI, USA.

National Engineering Research Center for Sugarcane, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China.

出版信息

Methods Mol Biol. 2020;2148:71-83. doi: 10.1007/978-1-0716-0623-0_4.

Abstract

Efficient and consistent chromosome identification is the foundation for successful cytogenetic studies. Fluorescent in situ hybridization (FISH) has been the most popular technique for chromosome identification in plants. Large insert genomic DNA clones, such as bacterial artificial chromosome (BAC) clones, and repetitive DNA sequences have been the most commonly used DNA probes for FISH. However, most of such traditional probes can only be used to identify a single chromosome or are too polymorphic to consistently identify the same chromosome in the target species. In contrast, FISH using oligonucleotide (oligo)-based probes is highly versatile. In this procedure, a large number of oligos specific to a chromosomal region, to an entire chromosome, or to multiple chromosomes are computationally identified, synthesized in parallel, and labeled as probes. In addition, each oligo probe can be used for thousands of FISH experiments and represents an infinite resource. In this chapter we describe a detailed protocol for amplification and labeling of oligo-based probes, relevant chromosome preparation, and FISH procedures.

摘要

高效且一致的染色体鉴定是成功进行细胞遗传学研究的基础。荧光原位杂交(FISH)已成为植物染色体鉴定最受欢迎的技术。大插入基因组 DNA 克隆,如细菌人工染色体(BAC)克隆和重复 DNA 序列,一直是 FISH 最常用的 DNA 探针。然而,大多数此类传统探针只能用于鉴定单个染色体,或者在目标物种中太多态,无法始终如一地鉴定同一染色体。相比之下,基于寡核苷酸(oligo)的探针的 FISH 具有高度通用性。在该程序中,大量针对染色体区域、整个染色体或多个染色体的特定寡核苷酸被计算识别、并行合成并标记为探针。此外,每个 oligo 探针可用于数千次 FISH 实验,代表了无限的资源。本章详细描述了基于寡核苷酸探针的扩增和标记、相关染色体制备和 FISH 程序。

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