The notion of sequencing the entire genome of a complex organism was not contemplated until 1986. The Nobel laureate Renato Dulbecco and others proposed at that time that the world scientific community be mobilized to undertake the sequencing of the entire human genome. One motive was to detect DNA damage in people who had survived the atomic bomb attacks and been exposed to radiation in Japan during World War II. But in order to detect changes in the human genome, scientists first needed to know its sequence.

The result was the publicly funded Human Genome Project, an enormous undertaking that was successfully completed in 2003. This effort was aided and complemented by privately funded groups. The genome project benefited from the development of many new methods that were first developed to sequence smaller genomes—those of prokaryotes and simple eukaryotes, the model organisms you have encountered in earlier chapters of this book. Many of these methods are still applied widely, and powerful new methods for sequencing genomes have emerged. These methods are complemented by new ways to examine phenotypic diversity in a cell’s proteins and in the metabolic products of the cell’s enzymes.

Many prokaryotes have a single chromosome, whereas eukaryotes have many. Because of their differing sizes, chromosomes are easy to separate from one another. You might think that the straightforward way to sequence a chromosome would be to start at one end and simply sequence the DNA molecule one nucleotide at a time. The task is somewhat simplified because only one of the two strands needs to be sequenced, the other being complementary. So if you have the sequence

5′ AAGCTCA.....3′

you know that the other strand must be

3′ TTCGAGT.....5′.

But sequencing a DNA molecule millions of bp long from one end to the other is just not possible using current methods. At most, about a few thousand bp can be sequenced at a time using current methods. The key to determining genome sequences is to break up long chromosomes into smaller DNA fragments and then thousands of fragments are sequenced individually at the same time.

In the 1970s Frederick Sanger and his colleagues invented a way to sequence DNA by using chemically modified nucleotides that were originally developed to stop cell division in cancer. This method, or a variation of it, was used to obtain the first human genome sequence as well as those of several model organisms. However, it was relatively slow, expensive, and labor-intensive. The first decade of the new millennium saw the development of faster and less expensive methods, often referred to under the general term high-throughput sequencing. These methods use miniaturization techniques first developed for the electronics industry, as well as the principles of DNA replication, often in combination with the *polymerase chain reaction (PCR).

High-throughput sequencing methods are rapidly improving. Just one of the many approaches is outlined here and illustrated in Figure 17.1. First the DNA is prepared for sequencing by attaching it to a solid surface and amplifying the DNA by PCR (see Figure 17.1A):

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Once the DNA has been attached to a solid substrate and amplified, it is ready for sequencing (see Figure 17.1B):

The power of this method derives from the fact that:

Sequencing millions of short DNA fragments is only part of the process of constructing the genome. Once these sequences have been determined, they need to be put together in the correct order. That is, they need to be arranged to reflect the sequence of the chromosomes from which they came. Imagine if you cut out every word in this book (there are more than half a million of them), put them on a table, and tried to arrange them in their original order! The enormous task of determining DNA sequences is possible because the original DNA fragments are overlapping.

Let’s illustrate the process using a single 10-bp DNA molecule. (This is a double-stranded molecule, but for convenience we show only the sequence of one strand.) The molecule is cut three ways (for example, using three different restriction enzymes). Cutting with the first enzyme generates the fragments:

TG, ATG, and CCTAC

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Cutting the same molecule with the second enzyme generates the fragments:

AT, GCC, and TACTG

Cutting with the third enzyme results in:

CTG, CTA, and ATGC

Can you put the fragments in the correct order? (The answer is ATGCCTACTG.) For genome sequencing, the sequence fragments are called “reads” (Figure 17.2). Of course, the problem of ordering 2.5 million fragments from human chromosome 1 (246 million bp) is more challenging than our 10-bp example above! The field of bioinformatics was developed to analyze DNA sequences using sophisticated mathematics and computer programs to handle the large amounts of data generated in genome sequencing.