INTRODUCTION TO THE TECHNIQUES OF MOLECULAR-BIOLOGY

The purpose of this chapter is to outline some of the common recombinant DNA methods in use today. These techniques are usually employed to isolate a defined portion of the genome, mostly a gene, from an organism or tissue of interest and, thereafter, to characterize the structure and function of this genetic material. To isolate a gene, genomic DNA is extracted from a selected tissue. For a better handling the relatively large DNA molecules are cut into a mixture of fragments by restriction endonucleases. The fragments are then separated from each other according to their size by gel electrophoresis. A procedure called Southern blotting is used to verify the presence of the desired gene in one of the DNA fragments separated on an agarose gel. The DNA fragments are transferred from the gel to a filter whereby the original fragment pattern is maintained. Then, a single-stranded DNA or RNA probe specific for the gene to be isolated is hybridized to its target fragments fixed to the filter. A radioactive or fluorescent tag is attached to the probe for subsequent identification. In cases where only transcribed sequences are to be isolated cytoplasmic messenger RNA (mRNA) is prepared instead of DNA. Analysis of RNA by a technique similar to Southern blotting is termed Northern blotting. Preservation of DNA sequences is usually achieved by DNA cloning. DNA cloning involves the insertion of a DNA fragment into a DNA vector and the stable incorporation of the recombinant DNA into a suitable host. Propagation of the host facilitates the amplification of the recombinant DNA for subsequent analysis. Reverse transcription into cDNA is a prerequisite for the cloning of RNA sequences. DNA sequencing either employs nucleotide-specific chemical degradation or the dideoxy nucleotide procedure. The generation of a genomic or cDNA library provides a collection of clones that can be stored indefinitely and used as a renewable source for DNA sequences. The polymerase chain reaction facilitates simple and rapid amplification of nucleic acid sequences without the necessity to use laborious DNA cloning procedures. While only touching upon a small number of recombinant DNA techniques, the reader can see the wide application of these methods.