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Molecular Basis of Heredity: Part 4. Gene Identification and Tests

Author(s): Raye L. Alford, PhD

Restriction Enzymes I

Restriction enzymes are enzymes produced by bacteria that recognize specific DNA sequences and cut the DNA strand at those sequences. Restriction enzymes can be used to cut DNA for a variety of purposes.

Restriction enzymes can be used to digest the genomic (chromosomal) DNA of an organism and generate a set of DNA fragments of manageable size. For example, digested DNA can be run on electrophoresis gels for evaluation of banding patterns. Variations in the banding patterns (pattern of restriction digestion) of certain genes between individuals affected by a genetic disease and those not affected may indicate the presence of an underlying molecular genetic alteration associated with the disease. Restriction analysis can also be used to detect polymorphic sites that, while not associated with disease, still interrupt the ability of a restriction enzyme to cut a particular segment of DNA. Restriction enzyme digestion of polymorphic sites and evaluation of the banding patterns that resulted were the basis for early DNA fingerprinting methods. Today, it is more common to find forensic DNA technology using length variations in repetitive elements to identify and distinguish among individuals because repetitive sequences can be more polymorphic (have more forms) than restriction fragments (gain or loss of restriction digestion) and have greater power to distinguish among individuals.

In the creation of DNA libraries, the DNA of an organism is digested (cut into fragments) with a restriction enzyme and cloned into vectors (bacteriophage, plasmid, cosmid) for further evaluation in the laboratory. Vectors are small, independently replicating DNA molecules carried by viruses or bacteria that can be manipulated in the laboratory to carry, and copy, genes of interest to researchers. Libraries can also be constructed using RNA as the starting material to synthesize complementary DNA molecules. In this case, a library called a cDNA library is created. Because cDNA libraries are based on RNA (transcribed genes) rather than genomic (total cellular) DNA, they are enriched for expressed (transcribed) genes whereas genomic DNA libraries contain all (or most of) the genes from an organism regardless of whether they were expressed (transcribed). Separate cDNA libraries from various individual cell types can be investigated to discover differences in gene expression between different types of tissues.