Most current methods of manipulating DNA, RNA, and proteins rely on prior knowledge of the nucleotide sequence of the genome of interest. But how were these sequences determined in the first place? And how are new DNA and RNA molecules sequenced today? In the late 1970s, researchers developed several strategies for determining, simply and quickly, the nucleotide sequence of any purified DNA fragment. The one that became the most widely used is called dideoxy sequencing or Sanger sequencing (Panel 8–1). This method was used to determine the nucleotide sequence of many genomes, including those of E. coli, fruit flies, nematode worms, mice, and humans. Today, cheaper and faster methods are routinely used to sequence DNA, and even more efficient strategies are being developed (see Panel 8–1). The original “reference” sequence of the human genome, completed in 2003, cost over $1 billion and required many scientists from around the world working together for 13 years. The enormous progress made in the past decade makes it possible for a single person to complete the sequence of an individual human genome in less than a day.
The methods summarized in Panel 8–1 for rapidly sequencing DNA can also be applied to RNA. Although methods are being developed to sequence RNA directly, it is most commonly carried out by converting the RNA to complementary DNA (using reverse transcriptase) and using one of the methods described for DNA sequencing. It is important to keep in mind that although genomes remain the same from cell to cell and from tissue to tissue, the RNA produced from the genome can vary enormously. We will see later in this chapter that sequencing the entire repertoire of RNA from a cell or tissue (known as deep RNA sequencing, or RNA-seq) is a powerful way to understand how the information present in the genome is used by different cells under different circumstances. In the next section, we shall see how RNA-seq has also become a valuable tool for annotating genomes.
To Be Useful, Genome Sequences Must Be Annotated
Long strings of nucleotides, at first glance, reveal nothing about how this genetic information directs the development of a living organism—or even what types of DNA, protein, and RNA molecules are produced by a genome. The process of genome annotation attempts to mark out all the genes (both protein-coding and noncoding) in a genome and ascribe a role to each. It also seeks to understand more subtle types of genome information, such as the cis-regulatory sequences that specify the time and place that a given gene is expressed and whether its mRNA undergoes alternative splicing to produce different protein isotypes. Clearly, this is a daunting task, and we are far short of completing it for any form of life, even the simplest bacterium. For many organisms, we know the approximate number of genes, and, for very simple organisms, we understand the functions of about half their genes.
In this section, we discuss broadly how genes are identified in genome sequences and what clues we can discern about their roles from simply inspecting their sequences. Later in the chapter, we turn to the more difficult problem of experimentally determining gene function.
How does one begin to make sense of a genome sequence? The first step is usually to translate in silico the entire genome into protein. There are six different reading frames for any piece of double-stranded DNA (three on each strand). We saw in Chapter 6 that a random sequence of nucleotides, read in frame, will
PANeL 8–1: DNA Sequencing Methods