Over the next decade, as molecular biologists tackle the task of sequencing the human genome on a massive scale, any number of innovations can be expected in mapping and sequencing technologies. But several of the central tools of molecular genetics are likely to stay with us -- much improved perhaps, but not fundamentally different. One such tool is the class of DNA-cutting proteins known as restriction enzymes. These enzymes, the first of which were discovered in the late 1960s, cleave double-stranded DNA molecules at specific recognition sites, usually four or six nucleotides long. For example, a restriction enzyme called EcoRI recognizes the single-strand sequence GAATTC and invariably cuts the double helix as shown in the illustration on the right.

Digesting DNA (36k GIF)

When digested with a particular restriction enzyme, then, identical segments of human DNA yield identical sets of restriction fragments. On the other hand, DNA from the same genomic region of two different people, with their subtly different genomic sequences, can yield dissimilar sets of fragments, which then produce different patterns when sorted according to size.

This leads directly to discussion of a second essential tool of modern molecular genetics, gel electrophoresis, for it is by electrophoresis that DNA fragments of different sizes are most often separated. In classical gel electrophoresis, electrically charged macromolecules are caused to migrate through a polymeric gel under the influence of an imposed static electric field. In time the molecules sort themselves by size, since the smaller ones move more rapidly through the gel than do larger ones. In 1984 a further advance was made with the invention of pulsed-field gel electrophoresis, in which the strength and direction of the applied field is varied rapidly, thus allowing DNA strands of more than 50,000 base pairs to be separated.

A third necessary tool is some means of DNA "amplification." The classic example is the cloning vector, which may be circular DNA molecules derived from bacteria or from bacteriophages (viruslike parasites of bacteria), or artificial chromosomes constructed from yeast or bacterial genomic DNA. The characteristic all these vectors share is that fragments of "foreign" DNA can be inserted into them, whereby the inserted DNA is replicated along with the rest of the vector as the host reproduces itself. A yeast artificial chromosome, or YAC, for instance, is constructed by assembling the essential functional parts of a natural yeast chromosome -- DNA sequences that initiate replication, sequences that mark the ends of the chromosomes, and sequences required for chromosome separation during cell division -- then splicing in a fragment of human DNA. This engineered chromosome is then reinserted into a yeast cell, which reproduces the YAC during cell division, as if it were part of the yeast's normal complement of chromosomes. The result is a colony of yeast cells, each containing a copy, or clone, of the same fragment of human DNA. One of the important achievements of the Human Genome Project has been to establish several libraries of such cloned fragments, using several different vectors (bacterial artificial chromosomes, P1 phages, and P1-derived cloning systems), that cover the entire human genome.

Spelling out the answer (59k GIF)

Another way of amplifying DNA is the polymerase chain reaction, or PCR. This enzymatic replication technique requires that initiators, or PCR primers, be attached as short complementary strands at the ends of the separated DNA fragments to be replicated. An enzyme then completes the synthesis of the complementary strands, thus doubling the amount of DNA originally present. Again and again, the strands can be separated and the polymerase reaction repeated -- so effectively, in fact, that DNA can be amplified by 100,000-fold in less than three hours. As with cloning vectors, the result is a large collection of copies of the original DNA fragment.

When a clone library can be ordered -- that is, when the relative positions on the human chromosomes can be established for all the fragments -- one then has the perfect resource for achieving the project's central goal, sequencing the human genome. How the sequencing is actually done can be illustrated by the most popular method in current use, the Sanger procedure, which is depicted schematically above. The first step is to prime each identical DNA strand in a preparation of cloned fragments. The preparation is then divided into four portions, each of which contains a different reaction-terminating nucleotide, together with the usual reagents for replication. In one batch, the replication reaction always produces complementary strands that end with A; in another, with G; and so on. Gel electrophoresis is used to sift the resulting products according to size, allowing one to infer the exact nucleotide sequence for the original DNA strand.

To Know Ourselves was prepared at the request of the U.S. Department of Energy, Office of Health and Environmental Research, as an overview of the Human Genome Project.