Pechhold et al. 2009. Transcriptional analysis of intracytoplasmically stained, FACS-purified cells by high-throughput, quantitative nuclease protection. Nature Biotechnol 27(11):1038–1043.Stiff DNA
Detection of target nucleic acid sequences by microarray analysis depends on their hybridization to complementary DNA probes affixed to defined spots on a surface. While this typically requires fluorescence labeling of the target molecules, there have been several recent attempts to exploit nanotechnology to enable label-less measurement of hybridization. In a recent Nature article, Husale et al. describe their new technique using atomic force microscopy (AFM) for the nanomechanical detection of changes in the stiffness of DNA probe molecules on a microarray surface before and after hybridization. They first validated their technique by hybridizing unlabeled target DNA to DNA oligonucleotide probes attached to the surface of a gold-coated silicon chip and then imaging the array by tapping-mode AFM. As the AFM cantilever tip contacts the DNA molecules on the surface, the measured interaction forces increase proportionally to local stiffness. Based on their observed force curves, the surface of the single-stranded DNA (ssDNA) region is stiffer than double-stranded DNA (dsDNA), which is due to the flatter conformation of ssDNA that causes the tip to “feel” the stiffness of the gold substrate. The difference in stiffness clearly differentiates dsDNA from ssDNA, and a computer-generated binary image permits the hybridized target molecules to be individually counted. Target DNA from 1 nM to 1 aM can be detected, which represents a 3- to 8-order magnitude increase when compared to the sensitivity of other methods of direct detection. RNA-DNA hybrids exhibited the same mechanical properties as dsDNA. In a test of their technique using complex biological samples, total RNA from colon and bladder tumors was assayed for two different microRNAs and the authors found the same pattern of differential expression in these two types of tumors observed in previous studies. Additionally, this pattern was clearly observed using as little as 0.2 ng of total RNA, a major reduction compared to conventional microarray methods which require at least 1 µg. This eliminates the need for the often difficult step of amplifying microRNAs when working with a limited amount of starting material. The ability to dispense with the labeling and enzymatic treatment of target nucleic acids, coupled with the specific and sensitive detection of hybridization in the attomolar range, makes this a very powerful new technique for microarray analysis.
Husale et al. DNA nanomechanics allows direct digital detection of complementary DNA and microRNA targets. Nature [Epub ahead of print, December 13, 2009, doi: 10.1038/nature08626].Promoter Profilers
Despite the importance of regulatory elements in gene expression, the details of their functional sequences have yet to be discerned. Saturation mutagenesis experiments, where a researcher generates and studies the impact of each possible mutation within a regulatory element, is one approach to accomplish this, but it has, thus far, only been applied in low-throughput experiments. In a recent article in Nature Biotechnology, Patwardhan et al. present a high-throughput method for the systematic analysis of mutations at each position in the bacteriophage promoters T3, T7, and SP6. The authors used a programmable microarray for the parallel synthesis of DNA oligonucleotides containing mutant promoters, each possessing a unique barcode sequence. After in vitro transcription of the library, RT-PCR and short-read sequencing was performed and transcriptional efficiencies of the promoters were determined based on the relative abundance of the associated barcodes. These data were normalized by comparison with the abundance of PCR-derived DNA barcodes to eliminate any variation from oligonucleotide concentrations in the initial library, and compared to the abundance of each barcode controlled by the native promoter as well as templates with random sequence in place of the native promoter. From the resulting transcriptional profiles, the authors were able to clearly define regions within the promoters where substitutions reduced transcription efficiency. In addition, they showed that it was possible to detect synergistic, compensatory, or antagonistic associations between multiple point mutations. The authors confirmed that their method worked well with mammalian transcriptional machinery by testing the cytomegalovirus, human beta globin gene, and human S100 calcium binding protein A4 promoters. Combined, these results indicate that synthetic saturation mutagenesis, with a quantitative readout using deep sequencing and cis-linked barcodes, reproducibly allows measurement of the relative activities of thousands of promoter variants in a single experiment.
Patwardhan et al. 2009. High-resolution analysis of DNA regulatory elements by synthetic saturation mutagenesis. Nature Biotechnol. 27(12):1173–1175.