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Stanford Team Develops DNA Attachment Method for Iterative Molecular Assays


NEW YORK (GenomeWeb) – Stanford researchers have developed a method of recovering genetic material for performing iterative testing. The group claims that the click-chemistry based tool can help save enough DNA for researchers to use in up to seven separate assays.

As researchers apply next-generation sequencing to identify infectious diseases, cancer, and other conditions, they typically struggle with collecting scarce amounts of target nucleic acids from samples. Limited amounts of material usually produce enough DNA or RNA for a single assay.

"When dealing with DNA derived from clinical samples like aspirates, cell-free DNA, or even material from single-cell assays, we wanted to have some way that we could re-interrogate the material, especially for downstream analysis," Stanford Genome Technology Center research engineer Billy Lau explained. "With this amount of limited material, it really restricts the type of experiments you can do."

In a proof-of-concept study published in Analytical Chemistry in January, Lau and Stanford professor Hanlee Ji showed that using click-based attachment of DNA sequencing libraries on agarose beads could allow repetitive primer extension assays for specific gene targets.

Lau explained that he explored several ways to conjugate the starting material in the study, finding that click chemistry was the easiest way to examine the DNA. The group selected cross-linked agarose beads because the material was amenable for most of the process.

"During PCR and other types of amplification, the [lack of material] could really skew or introduce some biases," Lau said. "When you're performing amplification, or any type of molecular assay, it's really hard to separate your original starting material from the product of that reaction." 

According to Lau, the method applies inverse electron demand Diels-Alder cycloaddition (IEDDA) to conjugate genomic DNA fragments tailed with TCO-modified nucleotides to an agarose solid substrate. The strand then generates a reusable DNA substrate for iterative polymerase-based enzymatic reactions. The study authors refer to the process as attachment-based primer extension (APEX).

Lau and Ji validated the APEX assay's performance for evaluating specific genetic alterations in both control and cancer reference standard DNA samples, targeting specific exon-flanking sequences in 185 genes.

They used a synthesized oligonucleotide primer pool – more than 12,000 unique primers – that bind to cancer-associated genomic regions sheared to about 500 base pairs. As validation, they tested the pool on normal DNA, a cancer standard from Horizon with known mutations previously verified by digital droplet PCR system, and genomic DNA from a matched tumor.

To begin the primer extension reaction, Lau and Ji incubated four spin columns containing the sample DNA, followed by two wash steps. The researchers sequenced the eluted fragments and then performed several sequence-read alignment and processing steps to evaluate the tool's performance.

They found that the overall representation of individual primers in the sequence data was relatively even, with more than 95 percent of target regions being within an order of magnitude of the median primer yield.

They then showed the stability of conjugated DNA libraries and related sequencing results using the same samples in multiple independent serial assays over a period of several months. They noted that the test samples therefore maintained integrity without degradation during the experiments.

Finally, Lau and Ji applied the method for storing and analyzing the patient-derived cancer DNA. They conjugated DNA libraries from a colorectal cancer (CRC) tumor sample to cross-linked agarose beads, and saw that multiplex primer extension produced similar performance to control samples.

In addition, they saw evidence of increased copy number of several exons such as NOTCH1, whose activation causes increased growth of CRC.

However, Lau noted that there were several limitations while developing the DNA recycling technology. One of the major issues was figuring out the correct conjugation chemistry. According to Lau, the researchers wanted a chemistry that required "mild" conditions and could work seamlessly with molecular biology.

"Click chemistry was something we wanted to employ from the start, but conventional 'azide-alkyne' click chemistry required the use of copper ions, an accelerating ligand, and special reaction conditions that made it difficult to employ," Lau explained. "The specific click chemistry method that we used (IEDDA) overcame this difficulty, as it is basically one-pot (meaning no other additional components), and can work in biological buffers."

In addition, Lau noted that IEDDA is much faster than standard click chemistry, which makes the tool amenable for working with analytes in the biomolecular concentration range.

Lau believes clinical researchers could use the tool to develop focused panels on certain genetic regions and iterate between the regions with the same samples. Users could also examine whole genome copy numbers in the samples, which Lau noted he is testing in a current study.  

While previous methods gave "a constraining amount of analyte material, you could use this conjugation to [instead] perform a whole gamut of assays without losing material," Lau said. "With this conjugation, [researchers] would be able to produce a comprehensive analysis of whatever targets that they might be looking for, whether it's detecting cancer or other types of early detection."

Lau also acknowledged that he still needs to validate the method and agarose bead material in further studies. However, he highlighted that the agarose beads in the proof-of-concept study had a very high binding capacity and are commercially available.  

"This means that we could quickly develop an assay to see if it would work or not, and down the road, after this proof of concept study has been established, we can start optimizing the type of materials or the solid phase we would use," Lau said.

In addition, Lau argued that the tool allows researchers to perform replicate testing in order to help confirm or improve certain results they might find.  If a researcher finds a variant of interest but doesn't understand its significance, they can repeat the assay over using the same genetic material to improve the low-frequency mutation's statistical confidence and determine if the result is relevant to the study.

While Lau noted that his group received a patent for the method in 2017, he declined to comment on any potential plans to commercialize the platform.

Since publishing the study, Lau said that his team has transitioned to using other materials like magnetic beads for DNA fragment collection. The team aims to push the boundaries of sample durability and improve further statistical confidence.

Lau said that he has several major goals in mind as the group develops the technology further: thoroughly validating the molecular retention of analytes, increasing the throughput (using liquid handling), expanding the type of analytes that researchers can potentially use (such as cell-free DNA and single-cell transcriptomes), and increasing the type of sequencing technologies researchers can apply (such as Oxford Nanopore's NGS platform.)

"You have to use your imagination, but really anything that passes the starting material without destroying it could potentially be compatible with that assay," Lau noted. "All the polymerase is doing is copying material as readout."