Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

ANALYZING AND MANIPULATING DNA
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With the possible exception of identical twins, the genome of each human differs
in DNA sequence from that of every other person on Earth. Using primer pairs
targeted at genome sequences that are known to be highly variable in the human
population, PCR makes it possible to generate a distinctive DNA fingerprint for
any individual (Figure 8–39). Such forensic analyses can be used not only to help
identify those who have done wrong, but also—equally important—to exonerate
those who have been wrongfully accused.
Both DNA and RNA Can Be Rapidly Sequenced
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