Protocol

Analyzing DNA Replication I: Labeling Animals, Tissues, and Cells with Bromodeoxyuridine (BrdU)

This protocol was adapted from "Analyzing DNA Replication: Nonisotopic Labeling," Chapter 13, in Basic Methods in Microscopy, (eds. Spector and Goldman). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2006.

INTRODUCTION

The number of cells traversing the cell cycle and the rate of progression through it provide important indices of cell growth and tumorigenicity. S-phase cells can also be identified by their high content of DNA polymerase and proliferating cell nuclear antigen, a component of the leading-strand polymerase. Although both these markers can be detected rapidly and conveniently using the appropriate antibodies, neither are found exclusively in S-phase cells. Immunolabeling after incorporation of modified DNA precursors (e.g., 5-bromodeoxyuridine [BrdU, bromodeoxyuridine]) allows more rapid and precise detection of cells in S-phase of the cell cycle. BrdU is phosphorylated by cells to give BrdUTP, and this precursor is incorporated into DNA instead of deoxythimidine triphosphate. In living cells, BrdU is incorporated into replication sites that can then be detected using fluorochrome or enzyme-coupled antibodies. Alternatively, DNA synthesis sites can be labeled at high resolution by incubating cells with analogs of the natural precursors of DNA. The cells are then fixed and the incorporation sites are detected using fluorochrome- or enzyme-tagged antibodies. Cells labeled in this way either in vivo or in vitro display a few hundred discrete nuclear sites early in S-phase, with distinct patterns of DNA replication that are characteristic of different stages of S-phase. "Foci" labeled after very short incubations correspond with sites where many replicons are duplicated simultaneously within massive protein complexes. This protocol provides details for incorporating DNA precursors into tissues in whole animals in vivo, isolated tissue in vitro, and cultured cells.