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Definition and discussion of genetic engineering

©2000 written by Gary Roberts, edited by Timothy Paustian, University of Wisconins-Madison

I C. DEFINITIONS AND DISCUSSION OF GENETIC ENGINEERING

I C1. GENERAL CONSIDERATIONS

The set of approaches grouped under the name "genetic engineering" differ from those of traditional genetics in a number of ways:

• Genetic engineering necessarily involves in vitro manipulations.

• As discussed in Other Types of Mapping, it deals with physical, rather than genetic, distances.

• It requires that you already have certain information (DNA sequences or position of restriction enzyme sites) for the relevant region of DNA.

• It requires that you physically isolate small but useful amounts of DNA from the region of interest.

• Most importantly, genetic engineering typically involves the characterization of the genotype prior to the analysis of the phenotype. This is sufficiently different from the rationale of traditional genetics that the term "inverse genetics" has been applied to genetic engineering. In traditional genetics, only "interesting mutants" (those with a detectably mutant phenotype) are analyzed: you do not receive positive data on regions which are not necessary for function. With site-directed mutagenesis, an interpretable result is obtained, positive or negative, with every mutant constructed and tested. This sort of scheme suffers in that it is a great deal of effort to build each mutant, which becomes especially painful if the majority do not produce an interesting phenotype.

Despite these differences, there are still a number of procedures and pitfalls shared by the two sets of approaches and this justifies their consideration in a single text. Their common ground is the belief that analysis of the phenotype caused by a non-wild-type genotype provides a unique insight into the system under study.

This text will treat these approaches in five sections: this section covers some of the definitions used; section IV F treats in vitro mutagenesis; section VI addresses the use of cloning methodologies in the construction of vectors; section IX A deals with the physical mapping and sequencing of DNA; and section IX pulls some of these tools together into integrated approaches for answering biological questions.

Given the rapidity of advances in these methodologies, specific techniques are not covered in detail.

I C2. DEFINITIONS

Since genetic engineering refers essentially to the in vitro manipulation of DNA, its application requires the ability to chemically isolate DNA from organisms. This is accomplished in three slightly different ways depending on the desired product. (a) Bulk DNA (all of the DNA in the cells of a culture typically including that of any plasmids) is isolated by breaking open the cells and purifying the DNA by precipitation and extraction, relying on those chemical properties of DNA which are different from that of other cell components. The product of such a treatment is a solution of reasonably large (greater than 10,000 bases in length, depending on the treatment) pieces of DNA where any given gene is present at a level appropriate to its frequency in the genome of the cell. (b) If you wish to isolate a particular gene or region exclusively, you typically need to place the desired region on a replicon (an autonomously replicating piece of DNA) other than the chromosome (a plasmid or a phage, as discussed in section VI). This provides an amplification of the amount of the desired region and also gives it physical properties (smallness and circularity, discussed in section VI) which allows it to be isolated readily from the rest of the DNA of the cell. DNA placed on such a replicon will often be referred to as cloned DNA. (c) Finally, the technique of PCR amplification allows the in vitro amplification of a region (see section IX A5).

Because cloned DNA can be purified in small but useful quantities, it is possible to chemically analyze the sequence of bases in that DNA. Such a sequence does not in itself answer biological questions but provides a powerful tool in the analysis of the cloned regions and the functions it encodes as will be described. Finally, the most crucial tools in the in vitromanipulation of DNA are restriction enzymes.

Restriction enzymes: To protect themselves from "invasion" by foreign DNA, many (all?) bacteria have developed enzyme systems that recognize specific sequences of bases and cut the DNA if that sequence has not been chemically modified. For our purposes, different restriction enzymes (a) recognize a 4-8 base sequence which may or may not involve contiguous bases; (b) generate either double-strand breaks (leaving "blunt" ends) or staggered single-strand ends (leaving short single-strand ends of one to several bases); (c) cut either in the recognized sequence or at a fixed distance to one side; (d) recognize sites which tend to be palindromes; (e) are commercially available with a wide range of specificities. These are general properties; a given enzyme will be quite specific in its recognized sequence and the position and nature of its endonucleolytic cuts. Digestion of DNA with a restriction enzyme produces various length fragments appropriate to the location of sites recognized by that enzyme.

Ligation: If fragments of DNA from different sources have been produced using a restriction enzyme that produces single-strand ends, these fragments can be annealed to each other using the single-strand regions since they are necessarily complementary. The two remaining single-strand nicks in the phosphate backbone are then repaired by an enzyme known as DNA ligase. The result is the generation of a single piece of DNA composed of segments originally from different sources. "Blunt end" ligation refers to the enzymatic fusion of DNA fragments lacking single- strand ends.

Labeling: In the definitions and techniques described below, a piece of nucleic acid is often referred to as "labeled". Until recently, this would almost necessarily mean that the sample was radioactive, allowing detection with X-ray film or scintillation counting. Recently, such "tagging" has been accomplished chemically, for example, by attaching biotin molecules randomly to the target molecules and eventually detecting the "label" by biotin-specific antibody linked to an easily assayed enzyme.

Hybridization: This refers to the annealing of single-stranded nucleic acid fragments to form a double-stranded duplex. Depending on the conditions (salt, temperature, etc.) under which the hybridization is performed, the amount of base mismatch can range from near zero ("high stringency") to 30-40% ("low stringency").

Library: The generation of a large set of vectors (phage or plasmid), each of which contains a piece (of variable size, 1-35 Kb, depending on the vector) of DNA from the organism of choice. Typically, such pieces are produced using restriction enzymes in such a way that a set of overlapping fragments is found in the library.

Southern analysis: Chromosomal (or any other DNA source) DNA is digested to completion (all possible nuclease cuts are made) with a given restriction enzyme and the DNA is subjected to electrophoresis in an agarose gel where it separates largely according to size. This pattern of DNA "bands" is transferred to a filter and treated such that the DNA will not come off and yet it is still substantially available for hydrogen bonding with complementary nucleic acid sequences. The treated filter is allowed to come in contact with labeled "probe" (DNA or RNA of the desired sequence) and then washed under conditions where properly double-stranded nucleic acid is stable but unhybridized probe is washed off. The position of hybridized probe then reflects the location of chromosomal sequences homologous to that probe.

Homologous: Two genes are said to be homologous if it is clear from their sequence that they are evolutionarily related. They cannot therefore be "highly" or "significantly" homologous, though their sequences can be "highly similar", or "very similar" etc. If genes are homologous, it does notfollow that the functions of the gene products are identical (or even necessarily very similar), though I'd bet that the gene products are structurally related.

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