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GE Researchers Plan to Sequence Single DNA Molecules Using “Closed Complex” Chemistry Soon

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By Julia Karow

Scientists at General Electric have demonstrated proof of concept for several aspects of “closed complex” sequencing, a single-molecule sequencing-by-synthesis chemistry under development at the firm for several years, and are planning to generate their first sequence data within three months, according to a company researcher.

The method will sequence DNA in a stepwise fashion, capturing a single terminal-phosphate-labeled nucleotide inside a polymerase prior to its addition to DNA, imaging the label, and resuming the reaction by adding a buffer with divalent metal ions.

At the National Human Genome Research Institute’s Advanced DNA Sequencing Technology Development Meeting in Chapel Hill, NC, last week, John Nelson, a scientist at the GE Global Research Center in Niskayuna, NY, reported that he and his colleagues have successfully formed stable closed complexes between primed single DNA molecules, polymerase, and labeled nucleotides on a solid support in a microfluidic system. They are now working on optimizing reaction conditions and building a “breadboard” platform to sequence DNA.

GE’s research team won a two-year, $900,000 grant to develop the chemistry under the NHGRI’s “$1,000 Genome” program in 2006 (see In Sequence 1/2/2007), followed by a two-year, $1.34 million follow-on grant last year (see In Sequence 10/13/2009).

Nelson explained that when DNA polymerase is mixed with primed DNA, it forms a binary complex. When labeled nucleotides are added and the correct nucleotide binds, an open tertiary complex forms at first, but then the enzyme’s “fingers” clamp down and trap the nucleotide in a closed complex, which is stable in the absence of divalent metal ions. After washing away free nucleotides, the label can be imaged. The base incorporation reaction is then completed — and the label cleaved — by adding buffer containing divalent magnesium or manganese ions, and the cycle is repeated.

Similar to Life Technologies’ “Starlight” single-molecule technology, which also uses terminal-phosphate-labeled nucleotides, the “closed complex” chemistry would generate natural DNA and allow for degraded polymerase to be replaced with fresh enzyme, thus promising long read lengths.

But unlike Life Tech’s method, and somewhat more akin to Helicos’ single-molecule technology, it would sequence DNA base-by-base instead of in real time, making it easier to image many reads in parallel.

Using a Biacore platform, Nelson’s group showed that they can form stable binary and tertiary complexes of surface-bound DNA, polymerase, and unlabeled nucleotides in a flow, in the absence of divalent metal ions. When they added metal ions to the closed complexes, the polymerases incorporated the nucleotides as expected. In addition, they were able to strip the enzyme off and add new enzyme repeatedly.

When the researchers added base-labeled or phosphate-labeled nucleotides, closed complexes also formed. However these were somewhat unstable. Raising the temperature helped, but so far, the scientists have not been able to form very stable closed complexes with terminal-phosphate-labeled nucleotides. They are hoping to achieve this by using new types of polymerases, generated by mutagenesis, and by synthesizing new types of terminally labeled nucleotides.

At the same time, they are developing a “breadboard” platform with a heated microfluidic stage to image single DNA molecules. Within three months, Nelson said, “we are hoping to generate sequence data on single molecules on this platform.”

Nelson told In Sequence that will take additional proof-of-principle work before GE will decide whether — and how — it might want to commercialize the “closed complex” sequencing chemistry.

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