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9.1 Fragmenting DNA 317

The techniques and approaches developed by the public and private Human
Genome Projects also catalyzed efforts to sequence the genomes of many species
other than humans. By 2016, whole-genome sequences had been completed for more
than 8100 distinct species, revolutionizing study in many areas such as microbiol-
ogy and plant biology. The availability of genome sequences for these other organ-
isms in turn has important benefits for our understanding of the human genome
through the identification of genes and other DNA elements that are conserved across
evolutionary lines.
In this chapter, we describe the methods that scientists developed to determine
the sequence of the human genome. The general ideas behind genome sequencing
are in fact not very complicated. Genomic researchers first fragment the genome into
much smaller pieces, and then isolate and amplify (that is, clone) individual pieces
by making so-called recombinant DNA molecules. Next, the scientists determine the
DNA sequence of individual purified, bite-sized fragments of the genome. Finally,
computer programs analyze the sequence of millions of these snippets to reconstruct
the sequence of the whole genome from which the pieces originated.

9.1 Fragmenting DNA well-defined cuts generate fragments suitable for manipu-
lation and characterization. A restriction enzyme recog-
nizes a specific sequence of bases anywhere within the
learning objectives genome and then severs two phosphodiester bonds at that
sequence, one in the sugar-phosphate backbone of each
1. Distinguish between digesting DNA with restriction strand. The fragments generated by restriction enzymes are
enzymes and mechanical shearing of DNA. referred to as restriction fragments, and the act of cutting
2. Describe how certain restriction enzymes generate DNA is often called digestion.
DNA fragments with sticky ends, while others generate Restriction enzymes originate in and can be purified
blunt-ended fragments. from bacterial cells. As explained in the Tools of Genetics
3. Calculate the average sizes and numbers of DNA Box Serendipity in Science: The Discovery of Restriction
fragments produced by digesting human genomic DNA Enzymes, these enzymes digest viral DNA to protect prokar-
with a given restriction enzyme. yotic cells from viral infection. Bacteria shield their own
4. Summarize the process by which gel electrophoresis genomes from digestion by these restriction enzymes
separates DNA fragments. through the selective addition of methyl groups (–CH 3 ) to
the restriction recognition sites in their genomic DNA. In
the test tube, restriction enzymes from bacteria recognize
Every intact diploid human cell, including the precursors of target sequences of four to eight base pairs (bp) in DNA
red blood cells, carries two nearly identical sets of 3 billion isolated from any other organism and cut the DNA at or
base pairs of information that, when unwound, extend 2 me- near these sites. Table 9.1 lists the names, recognition
ters in length. This is much too much material and informa- sequences, and microbial origins of just 10 of the close to
tion to study as a whole. To reduce its complexity, researchers 300 commonly used restriction enzymes.
first cut the genome into bite-sized pieces that can be ana- For the majority of these enzymes, the recognition
lyzed individually. One strategy to accomplish this goal is to site consists of four to six base pairs and exhibits a kind of
use enzymes to cut the genome at specific DNA sequences; palindromic symmetry in which the base sequences of
an alternative technique is to fragment the genome at ran- each of the two DNA strands are identical when read in
dom positions by shearing genomic DNA with mechanical the 5′-to-3′ direction. Because of this fact, base pairs on
forces. Both of these methods have their uses. either side of a central line of symmetry are mirror images
of each other. Each enzyme always cuts at the same place
relative to its specific recognition sequence, and most en-
Restriction Enzymes Cut zymes make their cuts in one of two ways: either straight
the Genome at Specific Sites through both DNA strands right at the line of symmetry to
produce fragments with blunt ends, or displaced equally
Researchers use restriction enzymes to cut the DNA re- in opposite directions from the line of symmetry by one
leased from the nuclei of cells at specific locations. These or more bases to generate fragments with single-stranded