使用脉冲场凝胶电泳分析染色体和染色体元件。

Using Pulsed-Field Gel Electrophoresis to Analyze Chromosomes and Chromosomal Elements.

作者信息

Pai Chen-Chun, Walker Carol, Humphrey Timothy C

机构信息

Department of Oncology, CRUK-MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, OX3 7DQ, United Kingdom.

出版信息

Cold Spring Harb Protoc. 2018 Apr 2;2018(4):pdb.prot092023. doi: 10.1101/pdb.prot092023.

DOI:10.1101/pdb.prot092023

PMID:28733409

Abstract

Pulsed field gel electrophoresis (PFGE) uses alternatively oriented pulsed electrical fields to separate large DNA molecules. Here, we describe PFGE protocols and conditions for separating and visualizing chromosomes between 0.5 and 6 Mb (optimal for analyzing the endogenous fission yeast chromosomes of 5.7, 4.6, and 3.5 Mb), and for shorter chromosomal elements of between 50 and 600 kb, such as the 530 kb Ch minichromosome. In addition to determining chromosome size, this technique has a wide range of applications, including determining whether DNA replication or repair is complete, defining the molecular karyotype of cells, analyzing chromosomal rearrangements, assigning genes or constructs to particular chromosomes, and isolating DNA from specific chromosomes.

摘要

脉冲场凝胶电泳（PFGE）使用交替定向的脉冲电场来分离大的DNA分子。在此，我们描述了PFGE的方案和条件，用于分离和可视化0.5至6兆碱基之间的染色体（最适合分析大小为5.7、4.6和3.5兆碱基的内源性裂殖酵母染色体），以及50至600千碱基之间较短的染色体元件，如530千碱基的Ch微型染色体。除了确定染色体大小外，该技术还有广泛的应用，包括确定DNA复制或修复是否完成、定义细胞的分子核型、分析染色体重排、将基因或构建体定位到特定染色体以及从特定染色体中分离DNA。