15.1: Introduction

Let’s start chapter by looking at technologies that led to genetic engineering. The ability to make recombinant DNA is such a seminal technology that just realizing it could be done and then doing it in a test tube for the first time earned Paul Berg a share of the 1980 Nobel Prize in Chemistry. First, we’ll look at cDNA synthesis (the synthesis of DNA copies of RNA), something retroviruses routinely do as part of their reproductive pathway (Figure. 15.1).

Figure 15.1: Infection by a retrovirus (left) requires reverse transcriptase encoded in its own genome to make a copy (cDNA) of its genome leading to replication of viral cDNA and the reproduction of new viruses in the infected cell (right).

As shown, the retrovirus injects its RNA into a target cell. There it transcribes a reverse transcriptase. This enzyme reverse-transcribes a copy DNA (cDNA) complementary to the viral RNA. The same enzyme next makes a (ds)cDNA (double stranded cDNA), which then replicates. These cDNAs are transcribed into new viral RNA genomes and mRNAs for viral proteins. The latter encapsulate the RNA genomes into new viruses. Reverse transcriptases, along with many viral, bacterial, and even eukaryotic enzymes and biomolecules, are now part of our recombinant DNA and genetic engineering toolkit.

We will see how a cDNA library is made and screened for a cDNA clone, and how a cloned cDNA can fish an entire gene out of a genomic library. Then we look at PCR (the polymerase chain reaction ), and how it produces (amplifies) millions of copies of a single gene (or other DNA sequence) from as little DNA as is found in a single cell. In addition to its well-publicized use in forensics and more recently in definitive tests for the SARS-Cov-2 viruses, PCR is also a vital laboratory tool for fetching, amplifying, and studying sequences of interest. These venerable technologies illustrate important principles of cloning and sequence analysis. Of course, the analysis of traditionally cloned and amplified DNA sequences has been used to study the evolution and expression of individual genes.

A cautionary note: despite the realized and future promises of such powerful tools to clone recombinant cDNAs and genomic DNA, we are sometimes misled! For example, knowing that a genetic mutation is associated with an illness usually leads to a search for how the mutation might cause the illness. But as researchers in any discipline keep warning us, correlation is not causation! In fact, we know that many phenotypes, including genetic disease, are not the result of a single mutant gene. Autism is just one example. Nevertheless, newer fields of genomics and proteomics leverage a growing battery of tools to study many genes and their regulatory networks at the same time. The molecular networking made possible by genomics and proteomics (and other colorful, holistic terms we’ll discuss later) promise to get us past naive and often incorrect notions of causation. We may soon be able to identify many correlations that might add up—if not to causation—at least to a propensity for genetic illness. We’ll look at some of these tools of leverage!

257 Overview of DNA Technologies

Learning Objectives

When you have mastered the information in this chapter, you should be able to do the following:

1. Design a molecular experiment using cDNA or a PCR product to clone a gene?
2. Determine when to make or to use a cDNA library or a genomic library.
3. Outline an experiment to purify rRNA from eukaryotic cells.
4. Outline an experiment to isolate and then clone human cDNA for further study.
5. Explain why (you might want to clone and to express a human growth hormone gene.
6. List components needed to make a cDNA library, using purified poly(A) RNA.
7. List the components needed to make a genomic library from isolated genomic DNA.
8. Compare PCR and genomic cloning as strategies for isolating a gene.
9. Outline a strategy for using fly DNA to obtain copies of a human DNA sequence.
10. Ask a research question that requires screening a genomic library for a specific gene.
11. Ask a question that requires using a microarray to obtain a gene you want to study.