DNA Sequencing in a Snap

An innnovative approach could target hard-to-sequence areas.

A novel sequencing technology being developed by a Massachusetts startup allows scientists to take photographs of the sequence of a DNA molecule. William Glover, president of ZS Genetics, based in North Reading, MA, says that his approach will allow scientists to read long stretches of DNA, enabling the sequencing of hard-to-read areas, such as highly repetitive regions in plants and some parts of the human genome. Longer sequences also allow scientists to distinguish between maternal and paternal chromosomes, which might have important diagnostic applications.

Seeing the sequence: This image shows a cluster of DNA molecules (black strands) that have been synthesized using bases that are specially labeled to be visible under an electron microscope. Scientists are using this technique to develop a novel sequencing technology.

Scientists at a recent sequencing conference in San Diego–where details of the technology were presented for the first time–were intrigued by the approach because it is totally different than even the newest methods on the market. “It’s surprising and potentially very powerful,” says Vladimir Benes, head of the Genomics Core Facility at the European Molecular Biology Laboratory, in Germany.

The cost of DNA sequencing has plummeted since a working draft of the human genome was completed in 2001. Most of the newest technologies currently in use generate very short sequences, about 30 to 150 base pairs, which are then stitched together with special software. But this method doesn’t always capture all the information in the genome, and some parts of the genome are difficult to sequence this way, says Glover.

ZS Genetics is a relative newcomer to the field and uses an approach vastly different than any other: electron microscopy. Glover predicts that by next year, the company’s technology will be able to generate readable lengths of DNA that are thousands of base pairs long, and he believes that ZS Genetics’ sequencing method will improve by a factor of 10 in the next couple of years, making the pieces even easier to assemble. The company was recently accepted as one of the teams in the Archon X Prize for Genomics, a $10 million award for the first privately funded team that can sequence 100 human genomes in 10 days.

“Any technology that can bring the read length to the 1,000 base-pairs range will definitely, at least for de novo sequencing, represent a major breakthrough,” says Benes. He says that the approach might be particularly useful for sequencing the genomes of plants, which often have highly complex genomes littered with repetitive sequences that are difficult to assemble computationally.

At a width of 2.2 nanometers, DNA is invisible under an average light microscope.

But electron microscopes, which detect the difference in charge between atoms, have a subnanometer resolution. While the sequence of natural DNA lacks enough contrast to be resolved with electron microscopy, Glover and his colleagues developed a novel labeling system to make the molecules more visible.

Researchers synthesize a new complementary strand of the molecule to be sequenced using bases–the letters that make up DNA–labeled with iodine and bromine. The labeled bases appear as either large or small dots under the electron microscope, allowing scientists to read the sequence. (Three different labels will be required to read the sequence of the four bases found in DNA. Three of the bases will have different labels; the fourth will simply remain unlabeled.)

The substrate on which the newly labeled molecules are imaged is made using semiconductor fabrication techniques. Scientists generate silicon wafers with an 11-nanometer-thick window, which is thin enough for the electron beam of the microscope to discern the DNA molecule from the substrate. ZS Genetics is also working on making even thinner wafers to boost resolution of the image.

DNA has a tendency to curl up into a tangled mass, so one of the biggest challenges has been untangling that ball into linear strands that can be read. Researchers first flow fluid through a microfluidic device with tiny channels. That device fits on top of the DNA-coated wafer. The force of the flow stretches the DNA molecules, which then stick to the silicon. An electron beam is shot through the wafer, and a camera captures the image from the other side. “The wafer is the major proprietary consumable,” says Glover. “It will be dirt cheap.”

When stretched, the DNA looks like a ladder with the bases forming the rungs. So far, the company has released images of a 23-kilobase piece of DNA using a single type of labeled base. Glover says that he and his team have also done multilabel sequencing, although he declined to give additional details.

Still, the technology has a ways to go before it’s market ready. “Lots of proof of principle methods can work in R&D, but bringing it to [market] is not trivial,” says Benes. Glover aims to have a prototype this summer that scientists can test, and a faster commercial system next year. He adds that because most of the system relies on existing technologies, it will be easy and inexpensive to upgrade the system with new cameras and software.

Longer reads will allow scientists to look at collections of genetic variations that have been inherited together, known as haplotypes. This kind of analysis can determine if a particular genetic variation has been passed down from the individual’s mother or father. Recent research suggests that in some cases, maternal or paternal inheritance can impact the severity of the disease, a phenomenon that may be more common than previously thought.

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