General Aspects of DNA Isolation and Purification

Abstract

DNA isolation is an essential technique in molecular biology. Isolation of high-molecular weight DNA has become very important with the increasing demand for DNA fingerprinting, restriction fragment length polymorphism (RFLP), construction of genomic or sequencing libraries and PCR analysis in research laboratories and industry. Also, DNA isolation is the first step in the study of specific DNA sequences within a complex DNA population, and in the analysis of genome structure and gene expression. The quantity, quality and integrity of DNA will directly affect these results. DNA constitutes a small percentage of the cell material and is usually localized in a defined part of the cell In prokaryotic cells, DNA is localized in the nucleoid that is not separated from the rest of the cell sap by a membrane. In eukaryotic cells, the bulk of DNA is localized in the nucleus, an organelle that is separated from the cytoplasm by a membrane. The nucleus contains about 90% of the total cellular DNA, the remaining DNA is in other organelles like mitochondria, chloroplasts or kinetochores. In viruses and bacteriophages, the DNA is encapsulated by a protein coat, and constitutes between 30 and 50 percent of the total mass of the virion. In prokaryotic and eukaryotic cells, DNA constitutes only about 1% of the total mass of the cell. The approximate composition of rapidly dividing E. coli cells and human cells (HeLa) is presented in Table 1.