Faster, Cheaper, Better

Easier genetic sequencing could make personalized medicine a reality

Imagine that you have the flu. After spending a couple of days with a hacking cough, a blazing fever, and muscles aching to the core, you finally head to the clinic. There, your primary care physician takes notes as you list your symptoms. As your monologue of complaints grinds to a halt, she pulls a page from the middle of your chart and nods. “It’s just as I suspected. Your genes make you especially vulnerable to this year’s strain of flu virus,” she says.

After reviewing a summary of your unique genetic sequence, she continues, “I’d give you the standard flu drug, but you have a mutation that makes you unable to metabolize that medicine.” However, you’re in luck—your doctor adds that a new drug developed specifically for people with your genetic profile has just entered the market. As she hands you the prescription, you marvel at the wonders of modern medicine.

Does this scenario sound too good to be true? For the moment, it is. The current cost of sequencing your genome is beyond your insurance company’s willingness to pay—on the order of millions of dollars. And the sequencing process is months too slow to be useful against this year’s flu virus.

Methods now in the works could remove this roadblock over the next several years by making the sequencing process quicker and less expensive. Then scientists can get down to the business of designing medicines and care that is specific to each person’s genes.

Tried, true, tired

The method that researchers currently use to sequence the genomes of people and other organisms is the same one, give or take a few tweaks, that’s been in place for the past 3 decades. That method, created in the mid-1970s by Frederick Sanger of the Medical Research Council in Cambridge, England, starts with the isolation of long strings of double-stranded DNA from cells. Each string contains pieces called bases—chemical units that go by the names adenine, thymine, guanine, and cytosine. Researchers typically refer to the bases by their initials: A, T, G, and C.

Once researchers have separated DNA from the rest of a cell’s innards, they use vibrations, high-pressure jets of water, or other forces to break those strings in random places into tiny pieces of approximately the same size—about 1,000 bases long. The scientists place these pieces one by one into bacteria that, as they divide, replicate an introduced DNA chunk as they would their own chromosome. Such replication gives researchers plenty of copies of the DNA pieces to work with.

Next, within each fractured-DNA solution, researchers split the double-stranded material into single strands and add them to lab dishes or test tubes containing two ingredients: a protein called DNA polymerase and a supply of the four bases.

In cells, the protein normally crawls along single-stranded DNA and adds bases one at a time to make the second strand and thereby return the DNA to its double-stranded form. At each step, DNA polymerase adds the base that complements the one already in place in the single strand. A pairs with T, and G pairs with C.

In the lab’s sequencing setup, DNA polymerase does the same thing, but the mix of bases includes a few that researchers have made easily detectable by tagging them with radioactivity or fluorescence. Those altered bases also carry chemical groups that will, once within DNA, halt the addition of more bases.

The rebuilding DNA strand then occasionally incorporates one of the tagged, full-stop bases instead of its unaltered brother. By looking for signs of these tagged bases in pieces of various lengths, researchers can figure out where each of the base types—A, T, G, or C—lies in the DNA strand.

Each step along this pathway takes just a few minutes. However, the human genome is made up of more than 3 billion pairs of bases, notes project manager Jeffrey Schloss of the National Human Genome Research Institute, which is part of the National Institutes of Health in Bethesda, Md.

“Right now, it takes months and months for a very large operation to sequence a human genome,” Schloss says. He adds that deciphering a sequence, while minimizing errors, costs about $5 million. That amount pays for skilled technicians, expensive chemicals, automated machines, and sometimes high-power cameras to detect tiny light flashes.

Shedding light

To reduce the time and cost of gene sequencing, Schloss explains, NIH is funding the development of several new approaches. Some of these strategies miniaturize and streamline the Sanger process. Others take different routes to a person’s genetic sequence.

One of these novel approaches uses light to indicate the order of bases in a string of DNA.

Several years ago, Mostafa Ronaghi of Stanford University in Palo Alto, Calif., and his colleagues discovered that a chemical called pyrophosphate is released each time DNA polymerase adds a base to a single strand of DNA. Next, an enzyme normally present in cells uses the two phosphate ions in each pyrophosphate molecule to make adenosine triphosphate (ATP), a chemical in which cells store energy.

To take advantage of this activity, Ronaghi and his colleagues prepped a test tube with ready-to-replicate DNA and a chemical that uses ATP to generate light. They then added a solution containing only one base. If that base was the one that the DNA was ready to incorporate, the subsequent reaction released pyrophosphate and the solution glowed. If the base wasn’t right, the researchers washed it out and added other bases, one at a time, until the solution glowed again. A computer recorded the sequence detected.

Now, working with a company called Biotage, based in Uppsala, Sweden, the researchers are automating the technology and miniaturizing it to put on a tiny chip. Although the current system still takes months to read a human-size genome, Ronaghi says that he expects, within the next 3 years, to streamline the process to sequence a person’s genome in a single day. He estimates the cost for such a service would be about $10,000—still expensive, but much lower than current prices.

With further tweaks that the company is contemplating, he says, “we might even have the opportunity to get it down to $1,000.”

Schloss says that other teams, such as the group led by Stephen Turner of Protea Biosciences in Morgantown, W. Va., aim to take a slightly different approach to sequencing a person’s genome. Rather than having a single light flash indicate that DNA polymerase has added a base to a lengthening DNA strand, these scientists use chemical reactions that would generate a different color of light for each base. This method would make it possible for a color-reading device to quickly register each time that DNA polymerase added a base to the DNA strand.

Poking holes

While their developers expect these technologies to reach the market within a few years, others in earlier experimental stages might make DNA sequencing faster and cheaper still. A team of researchers led by Reza Ghadiri of the Scripps Research Institute in La Jolla, Calif., is building its method around tiny holes called nanopores.

Other researchers noticed in the early 1990s that an enzyme called alpha hemolysin pokes nanosize holes into the cell membranes of organisms that some bacteria have infected. Because each DNA base is slightly smaller than an alpha hemolysin-created pore and has a characteristic shape, Ghadiri and his colleagues reasoned that they might distinguish the bases on a DNA strand moving through membrane pores.

The scientists placed a salt solution on each side of the membrane. Then, they threaded a single strand of DNA through a nanopore. The researchers monitored the flow of salt ions traversing the hole as each of the DNA bases squeezed through in sequence.

In the team’s initial experiments, the DNA passed through the hole too quickly to let the researchers read out differences in ion flow.

The researchers have since developed several ways to avoid that problem. For example, the team recently placed chemical groups that act as stoppers on each end of the DNA strand to be analyzed. Rather than slipping out of the pore, says Ghadiri, the DNA strand moves back and forth. “It goes in and out like you’re playing the cello,” he explains. After observing numerous passes of a sample DNA strand through a pore, the team distinguished the order of the bases.

In more-recent experiments, Ghadiri’s team tested a method to control the movement of the DNA strand. The researchers added DNA polymerase to one end of a strand that’s threaded through the pore. The polymerase is too wide to enter the pore, so as the polymerase crawls along the single strand, adding bases, it pulls through the DNA at a measured pace.

Other teams are developing similar nanopore technology using holes mechanically drilled through silicon and other materials.

Ghadiri says that “the jury is still out” on whether biological nanopores, such as the one he’s developing, or those in synthetic materials will be better for sequencing genomes. While organisms such as bacteria could eventually be engineered to develop pores with advanced capabilities—such as generation of a different electrical response for each of the bases within a string of DNA—synthetic nanopores wouldn’t need care and feeding as an organism does.

Regardless of which pore material wins out, Ghadiri says that the technique could dramatically lessen the time and cost of genome sequencing. It doesn’t require expensive chemicals and equipment. Ghadiri estimates that sequencing a person’s genome using nanopores will eventually take only hours and will cost less than $1,000.

Dangling carrot

A new competition could speed the development of faster sequencing techniques. The X Prize Foundation, a Santa Monica, Calif.–based group, runs private competitions promoting projects ranging from low-cost space travel to the invention of ultraefficient cars. The foundation announced last October that it would award $10 million to the first group to develop a way to sequence at least 100 people’s genomes in 10 days at a cost of no more than $10,000 per genome.

Three teams with sequencing experience are already on the roster of competitors for the prize, says Laurence H. Kedes, scientific director of the X Prize for Genomics.

“The solution to this problem could involve technologies outside current activity and may come out of left field. We want to have a big-enough carrot out there” to draw more than the usual genomic researchers, Kedes says.

He notes that several other scientists from both academia and industry have expressed interest in the contest.

Schloss says that both the new X Prize and further government funding increase the odds that patients will eventually have a routine genetic sequence in their medical records. Even then, he says, researchers have much work to do before such information can guide a doctor’s care. Scientists still don’t understand the function of most genes in the human genome.

Sequencing a person’s genome “will be relatively easy,” Schloss says. “We will soon have the ability to collect genome sequences on individuals faster than we’ll be able to interpret them.”

From the Nature Index

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