Overview

Because the genes of all organisms consist of DNA, and because a gene's structure largely determines its functional properties, analysis of any particular gene demands the isolation and structural analysis of DNA. To manipulate DNA experimentally, or even to determine its base sequence, chromosomal DNA must be reduced to small fragments, which are then sorted, identified, copied, and stored for future use.

This chapter explains some basic principles of cutting DNA enzymatically into fragments, separating pieces of DNA or RNA by size, making DNA copies of RNA, identifying one particular piece of DNA or RNA, copying DNA, and establishing large collections of DNA fragments for storage and retrieval.

Cutting and Fractionating DNA

Restriction Enzymes

It is easy to isolate chromosomal DNA. However, chromosomes tend to be long, and, even after random breakage, the DNA isolated from chromosomes exists as intractably long pieces. It is convenient to cut DNA into appropriate-sized fragments with bacterial restriction endonucleases (often simply called restriction enzymes), which cut only at particular sites.

Bacteria protect themselves against viral infection by cutting foreign DNA with restriction enzymes, so called because they restrict foreign DNA's ability to invade the cell. The cell distinguishes its own DNA from foreign DNA by methylating certain bases shortly after replication, using modification enzymes. At least one strand of the bacterium's own DNA is modified and therefore resistant to restriction, whereas foreign DNA is not modified and therefore susceptible to restriction.