As budgets tighten, throughputs increase, and downstream protocols become more exacting, serious efforts are being made to make the most of nucleic acid sample preparation. Whether it’s different devices to gather clinical or potential bioterror samples, innovative methods to enrich and purify samples, better ways to mash them up, or efficient ways to move them about, researchers at Knowledge Foundation’s “Sample Prep” meeting, held recently in San Diego, had much to talk about.
A lot has been made lately of next- (second) generation sequencing, characterized as being faster, cheaper, and more massively parallel than Sanger sequencing. Yet next-gen sequencing generally suffers from relatively short DNA read lengths.
FLIR Systems is gearing up for next-next (third) generation sequencing. Many of these newer sequencers will require DNA inputs that are in the tens, if not hundreds, of kilobases in length, said senior laboratory scientist Milena Iacobelli Martinez.
Working under a grant from the Defense Threat Reduction Agency (DTRA) to develop technology for detecting biothreats, FLIR has created an automated sample-preparation device that isolates 20–50 kb DNA. The prototype uses a single disposable cartridge to input up to 1 mL samples, and it lyses spores, vegetative cells, and viruses by a combination of chemical and mechanical means. It’s “really a compromise” between using enough strength to break open the spores and yet not overshear the DNA, she pointed out.
DNA in the lysate then gets concentrated using Boreal Genomics’ SCODA technology that is integrated into the prototype. “Their technology allows high molecular weight DNA to be isolated and low molecular weight DNA to be rejected,” Martinez explained. “In the end you get the concentrated DNA sample in a low volume (about 50 microliters), free of PCR inhibitors that you find in environmental samples and in clinical samples as well.”
Shorter DNA is likely to compete with and perhaps saturate out the longer strands, she said. It’s critical to remove the low molecular weight DNA before it gets to the sequencer, otherwise “you’re really not utilizing the full potential of the long-read technology.”
By switching run parameters on the SCODA, the instrument can also be used to concentrate all the nucleic acids, says Martinez, “so that it can be used for next-gen sequencing as well.”
SCODA—which stands for synchronous coefficient of drag alteration—takes advantage of the fact that nucleic acids undergo a dramatic, nonlinear, physical change under an electric field. “This distinguishes them physically from other molecules: proteins, humic acid in soil, polysaccharides—whatever you might have that could be inhibitors of downstream analysis,” said Andre Marziali, Boreal Genomics’ president and CSO.
Instead of relying on the more traditional solid-phase extraction—binding to a surface, washing, and eluting—SCODA “is taking DNA in a gel and focusing it to a spot at the center.” Meanwhile inhibitors, even those with similar chemical properties, either pass out of the gel or don’t enter it at all.
In his talk, Dr. Marziali, who is also director of engineering physics at the University of British Columbia, focused on a new Boreal development: sequence-specific extraction. By encoding a specific sequence in the gel itself, the instruments can enrich for that target. Single-stranded DNA is run on a gel at the melting temperature of the target probe duplex—the target is slowed down, but does not stick for very long. “What we’re really doing is we’re driving hundreds of thousands of hybridizations that cumulatively cause a focusing force on the target.”
Among other opportunities, Dr. Marziali is looking toward medical diagnostics. A pathogen may be present in a few copies per milliliter of a clinical blood sample containing 100 µg of human DNA in the background, and “we believe that we can enrich for the pathogen DNA, leaving the human DNA behind,” Dr. Marziali said. Similarly, disease markers can be enriched in cancer patients’ blood, as can fetal DNA taken from a maternal sample—situations “where ultimately the inhibitors are not now blood components, they’re really human DNA.
“If we put a pool of DNA into our instrument that has, say, two sequences that differ by a single nucleotide, we can enrich our targeting against a single nucleotide mismatch by about 10,000 fold. In fact, we’re so specific that we can distinguish two sequences that differ by a single methylation site.”