Popular DNA purification methods work well for many applications, but often yield nothing but frustration in the presence of contaminants like humic acid that share affinity for the separation matrix. Pel et al., writing in the Proceedings of the National Academy of Sciences, put a new spin on this problem, in the form of rotating electric field electrophoresis, which leverages the unique response of nucleic acids to electric fields. Unlike most other molecules, the drift velocity of DNA responds nonlinearly to changes in field strength. By properly modulating the electric field, DNA dispersed evenly throughout a square agarose gel will become focused in its center, while contaminants will experience no net movement. In this more practical implementation, the sample is placed in a 5-mL loading reservoir and introduced into the gel by a direct current (DC) field. “Electrophoretic washing” is performed by refreshing the buffer in the reservoir, and during application of the rotating fields, maintaining a small DC bias in the direction of the loading chamber. Although negatively charged contaminants will enter the gel, they will not be trapped by the rotating fields and will exit with the washing field. The power of the technique is astounding: DNA from 200 bp to 1.6 Mbp can be concentrated, and consistent PCR amplification is possible from a 56-zM DNA solution (<200 molecules total). In DNA samples spiked with humic acid, the new method worked better than silica column or magnetic bead separation, and, impressively, the strategy enabled metagenomic analysis of unpurified oil sands samples. At present, the method demands patience (some purifications required 17-h runs), but future higher-voltage designs should improve speed dramatically. Other upcoming innovations are likely to allow isolation of RNA and denatured proteins.
Pel et al. 2009. Nonlinear electrophoretic response yields a unique parameter for separation of biomolecules. Proc Natl Acad Sci USA 106(35):14796–1480.Broken Record
Some of the most tantalizing sources of genomic information—bones from extinct creatures, archived clinical samples, and forensic material—are also the most technically challenging to sequence, thanks to limitations in DNA amount and purity. In this context, whole-extract shotgun sequencing has not proved efficient; in addition, targeted sequencing can be desirable for comparative genomics and mitochondrial analysis. Existing target enrichment procedures tend to be unsuitable for degraded and/or extremely limited amounts of DNA, but, as described in Genome Research, Stiller et al. introduce direct multiplex sequencing as a solution to this problem, showing their method's potential by sequencing the complete mitochondrial genomes of 31 specimens of extinct cave bear. Amazingly, despite prevailing views on the dangers of primer dimers, the method uses unoptimized multiplex PCR. For this project, the authors used two sets of 64 primer pairs to cover the mitochondrial genome; although each set comprised independent amplicons, together they were designed to produce overlapping reads. Subsequent magnetic bead–based purification (selecting fragments of ~100 bp or more) proved sufficient to remove nonspecific amplification products. Adapters with short sample-specific barcode sequences enabled the assignment of sequence to source after pooling the sequencing libraries. Sequencing was performed by parallel microemulsion pyrosequencing of bead-tethered amplicons, yielding a 96% complete mitochondrial genome set of sufficient quality to support phylogenetic inferences. Preliminary experiments with samples from other organisms showed similar amplification success rates, suggesting the rigorous size selection steps are generally sufficient to allow unoptimized multiplexed PCRs.
Stiller et al. Direct multiplex sequencing (DMPS)—a novel method for targeted high-throughput sequencing of ancient and highly degraded DNA. Genome Res [Epub ahead of print, September 2, 2009, doi:10.1101/gr.095760.109].Beyond the Surface
Methodological advances are sometimes won by questioning conventional wisdom. Such is the case with a paper from Moyroud et al., which appeared in The Plant Journal. It had been generally accepted that surface plasmon resonance (SPR) offered little to the study of transcription factor binding: since SPR interactions must occur within ~200 nm of the chip surface, it was perceived that the technique was restricted to relatively short DNA fragments. But Moyroud et al. hypothesized that the conformational properties of long DNA molecules would place the transcription factor–DNA binding event close enough to be detected. Experiments bore this out, with binding detected even for DNA fragments in excess of 3 kb. Importantly, the technique is equally capable of qualitatively flagging the presence of a binding site and making quantitative comparisons between wild-type and mutated recognition sites. Whole-promoter SPR should help confirm potential transcriptional regulatory interactions detected by microarray or chromatin immunoprecipitation, and should be particularly appropriate when candidate transcription factor binding sites are not picked up in silico. The technique is also well-suited for studying DNA-protein binding in a more biologically relevant context, such as cooperative binding of multiple sequences dispersed throughout a promoter region. Finally, given the increasing sophistication of SPR platforms, the technique may enable systems-level analysis of transcriptional networks.