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一种基于CRISPR/Cas9的现场标记方法

Bhanu Prakash Potlapalli¹, Takayoshi Ishii², Kiyotaka Nagaki³

¹Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Seeland, Germany.

Methods in molecular biology (Clifton, N.J.)

|June 19, 2023

概括

CRISPR-FISH是一种新的RNA引导内核酶在位标记 (RGEN-ISL) 方法,通过保留色素结构,克服了传统光在位杂交 (FISH) 的局限性. 这种技术可以在各种植物样本中精确地标记DNA.

科学领域:

分子生物学分子生物学
遗传学是一个遗传学.
生物技术是生物技术.

背景情况:

传统的光在位杂交 (FISH) 需要严厉的变性步骤,损害染色质结构.
在固定的样本中可视化特定的DNA序列对于遗传和细胞学研究至关重要.
现有的方法往往会损害样本的完整性.

研究的目的:

介绍和详细说明CRISPR-FISH (基于CRISPR/Cas9的现场标记) 的协议,也称为RNA引导内核酶现场标记 (RGEN-ISL).
展示CRISPR-FISH在不同植物物种中标记重复DNA序列的应用.
为结合CRISPR-FISH与免疫染色提供方法.

主要方法:

基于CRISPR/Cas9的现场标签协议的开发 (CRISPR-FISH/RGEN-ISL).
在酸上应用CRISPR-FISH:以乙醇或甲固定核,染色体和组织部分.
免疫染色技术与CRISPR-FISH的整合.

主要成果:

CRISPR-FISH有效地标记了各种植物物种中重复的DNA序列,而不会损害染色体.
协议可以适应不同的固定方法和样品类型 (核,染色体,组织部分).
证明了CRISPR-FISH与免疫染的成功组合.

结论:

在DNA序列可视化方面,CRISPR-FISH提供了一种比传统的FISH更温和,更有效的替代方案.
这种方法保留了染色质的完整性,允许进行更详细的下游分析.
CRISPR-FISH扩大了植物基因组学和细胞遗传学研究的工具包.

关键词:

这就是CRISPR-FISH.这就是CRISPR/Cas9的作用.染色体的动态染色体的动态细胞遗传学细胞遗传学干旱地区是干旱地区.鱼类鱼类是一种鱼类.分子细胞遗传学分子细胞遗传学植物染色体是植物的染色体.在RGEN-ISLL中,

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