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Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis. 7th edition. New York: W. H. Freeman; 2000.

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An Introduction to Genetic Analysis. 7th edition.

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Methodologies used in genetics

The study of genes has proved to be a powerful approach to understanding biological systems. Because genes affect virtually every aspect of the structure and function of an organism, being able to identify and determine the role of genes and the proteins that they encode is an important step in charting the various processes that underly a particular character under investigation. It is interesting that geneticists study not only hereditary mechanisms, but all biological mechanisms. Many different methodologies are used to study genes and gene activities, and these methodologies can be summarized briefly as follows:

1.

Isolation of mutants affecting the process under study. Each mutant gene reveals a genetic component of the process, and together they show the range of proteins that interact in that specific process.

2.

Analysis of progeny of controlled matings (“crosses”) between mutants and other discontinuous variants. This type of analysis identifies genes and their alleles, their chromosomal locations, and their inheritance patterns. These methods will be introduced in Chapter 2.

3.

Biochemical analysis of cellular processes controlled by genes. Life is basically a complex set of chemical reactions; so studying the ways in which genes are relevant to these reactions is an important way of dissecting this complex chemistry. Mutant alleles underlying defective function (see method 1) are invaluable in this type of analysis. The basic approach is to find out how the cellular chemistry is disturbed in the mutant individual and, from this information, deduce the role of the gene. The deductions from many genes are assembled to reveal the larger picture.

4.

Microscopic analysis. Chromosome structure and movement have long been an integral part of genetics, but new technologies have provided ways of labeling genes and gene products so that their locations can be easily visualized under the microscope.

5.

Analysis of DNA directly. Because the genetic material is composed of DNA, the ultimate characterization is the analysis of DNA itself. Many procedures, including cloning, are used. Cloning is a procedure by which an individual gene can be isolated and amplified (multiply copied) to produce a pure sample for analysis. After the clone of a gene has been obtained, its nucleotide sequence can be determined and hence important information about its structure and function can be obtained. Furthermore cloned genes can be used as biological probes, as described next.

Cloning will be fully described in Chapters 12, 13, and 14, but a brief overview is necessary here so that some concepts can be applied in earlier chapters. Cloning works by inserting the gene to be amplified into a small accessory chromosome and letting this chromosome do the job of replicating and amplifying its “passenger” fragment. This small chromosome is called a vector or carrier. Commonly used vectors are plasmids, which are nonessential extra DNA molecules found naturally in many bacteria. The DNA of any organism can be inserted into a vector; the hybrid construct is then introduced into a single bacterial cell and a large culture grows from this cell (Figure 1-16). The vector and insert can then be removed from disrupted cells and studied as appropriate.

Figure 1-16. Basic cloning methodology.

Figure 1-16

Basic cloning methodology.

Detecting specific molecules of DNA, RNA, and protein

Because the main macromolecules of genetics are DNA, RNA and protein, genetic analysis often requires the detection of specific molecules of each of these three types. How can specific molecules be identified among the thousands of types in the cell? The most extensively used method for detecting specific macromolecules in a mixture is probing. This method makes use of the specificity of intermolecular binding, which we have already encountered several times. The probe is labeled in some way, either by a radioactive atom or by a fluorescent compound, so that the site of binding can easily be detected. Let's look at probes for DNA, RNA, and protein.

Probing for a specific DNA

A cloned gene can act as a probe for finding segments of DNA that have the same or a very similar sequence. For example, if a gene G from a fungus has been cloned, it might be of interest to determine whether plants have the same gene. The use of a cloned gene as a probe takes us back to the principle of base complementarity. The probe works through the principle that, in solution, the random motion of probe molecules enables them to find and bind to complementary sequences. The experiment must be done with separated DNA strands, because then the bonding sites of the bases are unoccupied. DNA from the plant is extracted and cut with one of the many available types of restriction enzymes, which cut DNA at specific target sequences of four or more bases. The target sequences are at the same positions in all the plant cells used, so the enzyme cuts the genome into defined populations of segments of specific sizes. The fragments can be fractionated by using electrophoresis.

Electrophoresis fractionates a population of nucleic acid fragments on the basis of size. The cut mixture is placed in a small well in a gelatinous slab (a gel), and the gel is placed in a powerful electrical field. The electricity causes the molecules to move through the gel at speeds inversely proportional to their size. After fractionation, the separated fragments are blotted onto a piece of porous membrane, where they maintain the same relative positions. This procedure is called a Southern blot. After having been heated to separate the DNA strands and hold the DNA in position, the membrane is placed in a solution of the probe. The single-stranded probe will find and bind to its complementary DNA sequence. For example,

Image ch1e6.jpg

On the blot, this binding concentrates the label in one spot, as shown in Figure 1-17a.

Figure 1-17. Probing DNA, RNA, and protein mixtures.

Figure 1-17

Probing DNA, RNA, and protein mixtures.

Probing for a specific RNA

It is often necessary to determine whether a gene is being transcribed in some particular tissue. This determination can be accomplished by using a modification of the Southern analysis. Total mRNA is extracted from the tissue, fractionated electrophoretically, and blotted onto a membrane (this procedure is called a Northern blot). The cloned gene is used as a probe and its label will highlight the mRNA in question if it is present (Figure 1-17b).

Probing for a specific protein

Probing for proteins is generally performed with antibodies because an antibody has a specific lock-and-key fit with its antigen. The protein mixture is electrophoresed and blotted onto a membrane (this procedure is a Western blot). The position of a specific protein on the membrane is revealed by bathing the membrane in a solution of antibody obtained from a rabbit into which the protein has been injected. The position of the protein is revealed by the position of the label that the antibody carries (Figure 1-17c).

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2000, W. H. Freeman and Company.
Bookshelf ID: NBK22047

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