Chapter 6: Molecular Genetic Techniques

Chapter Introduction

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CHAPTER 6

Molecular Genetic Techniques

The mouse on the right has cataracts because it carries a single copy of a dominant mutant allele of the gene for gamma-crystallin. The mouse on the left, with normal eyes, was produced from a zygote in which the mutant allele was corrected back to the wild-type state by the CRISPR-Cas9 genome editing method.

[Jinsong Li.]

OUTLINE

6.1 Genetic Analysis of Mutations to Identify and Study Genes

6.2 DNA Cloning and Characterization

6.3 Using Cloned DNA Fragments to Study Gene Expression

6.4 Locating and Identifying Human Disease Genes

6.5 Inactivating the Function of Specific Genes in Eukaryotes

In the field of molecular cell biology, reduced to its most basic elements, we seek an understanding of the biological behavior of cells in terms of the underlying chemical and molecular mechanisms. Often the investigation of a new molecular process focuses on the function of a particular protein or set of proteins. There are three fundamental questions that cell biologists usually ask about a newly discovered protein: What is the function of the protein in the context of a living cell? What is the biochemical function of the purified protein? Where is the protein located? To answer these questions, investigators employ three molecular genetic tools: the gene that encodes the protein, a mutant cell line or organism that lacks the functional protein, and a source of the purified protein itself. In this chapter, we consider various aspects of two basic experimental strategies for obtaining all three tools (Figure 6-1).

FIGURE 6-1 Overview of two strategies for relating the function, location, and structure of gene products. A mutant organism is the starting point for the classical genetic strategy (green arrows). The reverse strategy (orange arrows) usually begins with identification of a protein-coding sequence by analysis of genomic sequence databases. In both strategies, the actual gene is isolated either from a DNA library or by specific amplification of the gene sequence from genomic DNA. Once a cloned gene is isolated, it can be used to produce the encoded protein in bacterial or eukaryotic expression systems. Alternatively, a cloned gene can be inactivated by one of various techniques and used to generate mutant cells or organisms.

The first strategy, often referred to as classical genetics, begins with isolation of a mutant that appears to be defective in some process of interest. Genetic methods are then used to identify and isolate the affected gene. The isolated gene can be manipulated to produce large quantities of the protein it encodes for biochemical experiments and to design probes for studies of where and when the protein is expressed in an organism. The second strategy follows essentially the same steps as the classical approach, but in the reverse order, beginning with isolation of an interesting protein or its identification based on analysis of an organism’s genomic sequence. Once the corresponding gene has been isolated, that gene can be altered and then reinserted into an organism. With both strategies, by examining the phenotypic consequences of mutations that inactivate a particular gene, geneticists are able to connect knowledge about the sequence, structure, and biochemical activity of the encoded protein to its function in the context of a living cell or multicellular organism.

An important component in both strategies for studying a protein and its biological function is isolation of the corresponding gene. Thus we discuss various techniques by which researchers can isolate, sequence, and manipulate specific regions of an organism’s DNA. Next we introduce a variety of techniques that are commonly used to analyze where and when a particular gene is expressed and where in the cell its protein is localized. In some cases, knowledge of protein function can lead to significant medical advances, and the first step in developing treatments for an inherited disease is to identify and isolate the affected gene, a process we describe here. Finally, we discuss techniques that abolish normal protein function in order to analyze the role of the protein in the cell.

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