Chapter 8 Isolation of Micronuclear and Macronuclear DNA

Abstract

This chapter discusses several methods for the isolation of micronuclei (MIC) and macronuclei (MAC). The chapter describes a procedure that uses Percoll gradients for purifying the nuclei, where separation of MIC from MAC occurs as a result of their difference in size. When examined under the light microscope, the purified MAC are usually contaminated with as little as one MIC per 200–500 MAC, with a yield around 30% of the total MAC available. The purified MIC are contaminated with as little as one MAC per 1000–2000 MIC, with an average yield of 30–40% of the total MIC in the cells harvested. Quantitative analyses of electron micrographs indicate a higher estimate of cross-contamination of the purified nuclei, which may be due to the real difficulty in identifying small MIC or MAC fragments. Smearing of the nuclear DNA in pulse-field gels suggests that some of the MAC DNA is degraded and that only a small percentage of the MIC DNA has a molecular weight greater than 1 Mb. For most purposes this DNA is adequate for analyses using conventional vectors and restriction digests, but it is not suitable for cloning large DNA fragments and for separation in pulse-field gels.