Nucleic Acids Research

Carter, Darren J.; Cary, R. Bruce

doi: 10.1093/nar/gkm269pmid: 17478499

Widely used nucleic acid assays are poorly suited for field deployment where access to laboratory instrumentation is limited or unavailable. The need for field deployable nucleic acid detection demands inexpensive, facile systems without sacrificing information capacity or sensitivity. Here we describe a novel microarray platform capable of rapid, sensitive nucleic acid detection without specialized instrumentation. The approach is based on a miniaturized lateral flow device that makes use of hybridization-mediated target capture. The miniaturization of lateral flow nucleic acid detection provides multiple advantages over traditional lateral flow devices. Ten-microliter sample volumes reduce reagent consumption and yield analyte detection times, excluding sample preparation and amplification, of <120 s while providing sub-femtomole sensitivity. Moreover, the use of microarray technology increases the potential information capacity of lateral flow. Coupled with a hybridization-based detection scheme, the lateral flow microarray (LFM) enables sequence-specific detection, opening the door to highly multiplexed implementations for broad-range assays well suited for point-of-care and other field applications. The LFM system is demonstrated using an isothermal amplification strategy for detection of Bacillus anthracis, the etiologic agent of anthrax. RNA from as few as two B. anthracis cells was detected without thermocycling hardware or fluorescence detection systems.

Morgan, Hugh P.; Estibeiro, Peter; Wear, Martin A.; Max, Klaas E.A.; Heinemann, Udo; Cubeddu, Liza; Gallagher, Maurice P.; Sadler, Peter J.; Walkinshaw, Malcolm D.

doi: 10.1093/nar/gkm040pmid: 17488853

We have developed a novel DNA microarray-based approach for identification of the sequence-specificity of single-stranded nucleic-acid-binding proteins (SNABPs). For verification, we have shown that the major cold shock protein (CspB) from Bacillus subtilis binds with high affinity to pyrimidine-rich sequences, with a binding preference for the consensus sequence, 5′-GTCTTTG/T-3′. The sequence was modelled onto the known structure of CspB and a cytosine-binding pocket was identified, which explains the strong preference for a cytosine base at position 3. This microarray method offers a rapid high-throughput approach for determining the specificity and strength of ss DNA–protein interactions. Further screening of this newly emerging family of transcription factors will help provide an insight into their cellular function.

Rivero-Müller, Adolfo; Lajić, Svetlana; Huhtaniemi, Ilpo

doi: 10.1093/nar/gkm250pmid: 17517785

Functional genomics require manipulation and modification of large fragments of the genome. Such manipulation has only recently become more efficient due to the discovery of different techniques based on homologous recombination. However, certain limitations of these strategies still exist since insertion of homology arms (HAs) is often based on amplification of DNA sequences with PCR. Large quantities of PCR products longer than 4–5 kb can be difficult to obtain and the risk of mutations or mismatches increases with the size of the template that is being amplified. This can be overcome by adding HAs by conventional cloning techniques, but with large fragments such as entire genes the procedure becomes time-consuming and tedious. Second, homologous recombination techniques often require addition of antibiotic selection genes, which may not be desired in the final construct. Here, we report a method to overcome the size and selection marker limitations by a two- or three-step procedure. The method can insert any fragment into small or large episomes, without the need of an antibiotic selection gene. We have humanized the mouse luteinizing hormone receptor gene (Lhcgr) by inserting a ∼55 kb fragment from a BAC clone containing the human Lhcgr gene into a 170 kb BAC clone comprising the entire mouse orthologue. The methodology is based on the rationale to introduce a counter-selection cassette flanked by unique restriction sites and HAs for the insert, into the vector that is modified. Upon enzymatic digestion, in vitro or in Escherichia coli, double-strand breaks are generated leading to recombination between the vector and the insert. The procedure described here is thus an additional powerful tool for manipulating large and complex genomic fragments.

Carr, Ian M.; Valleley, Elizabeth M. A.; Cordery, Sarah F.; Markham, Alexander F.; Bonthron, David T.

doi: 10.1093/nar/gkm330pmid: 17517768

Bisulphite genomic sequencing is a widely used technique for detailed analysis of the methylation status of a region of DNA. It relies upon the selective deamination of unmethylated cytosine to uracil after treatment with sodium bisulphite, usually followed by PCR amplification of the chosen target region. Since this two-step procedure replaces all unmethylated cytosine bases with thymine, PCR products derived from unmethylated templates contain only three types of nucleotide, in unequal proportions. This can create a number of technical difficulties (e.g. for some base-calling methods) and impedes manual analysis of sequencing results (since the long runs of T or A residues are difficult to align visually with the parent sequence). To facilitate the detailed analysis of bisulphite PCR products (particularly using multiple cloned templates), we have developed a visually intuitive program that identifies the methylation status of CpG dinucleotides by analysis of raw sequence data files produced by MegaBace or ABI sequencers as well as Staden SCF trace files and plain text files. The program then also collates and presents data derived from independent templates (e.g. separate clones). This results in a considerable reduction in the time required for completion of a detailed genomic methylation project.

Stafford, Phillip; Brun, Marcel

doi: 10.1093/nar/gkl1133pmid: 17478523

Microarray gene expression data becomes more valuable as our confidence in the results grows. Guaranteeing data quality becomes increasingly important as microarrays are being used to diagnose and treat patients (1–4). The MAQC Quality Control Consortium, the FDA's Critical Path Initiative, NCI's caBIG and others are implementing procedures that will broadly enhance data quality. As GEO continues to grow, its usefulness is constrained by the level of correlation across experiments and general applicability. Although RNA preparation and array platform play important roles in data accuracy, pre-processing is a user-selected factor that has an enormous effect. Normalization of expression data is necessary, but the methods have specific and pronounced effects on precision, accuracy and historical correlation. As a case study, we present a microarray calibration process using normalization as the adjustable parameter. We examine the impact of eight normalizations across both Agilent and Affymetrix expression platforms on three expression readouts: (1) sensitivity and power, (2) functional/biological interpretation and (3) feature selection and classification error. The reader is encouraged to measure their own discordant data, whether cross-laboratory, cross-platform or across any other variance source, and to use their results to tune the adjustable parameters of their laboratory to ensure increased correlation.

Mrázek, Jan; Kreutmayer, Simone B.; Grässer, Friedrich A.; Polacek, Norbert; Hüttenhofer, Alexander

doi: 10.1093/nar/gkm244pmid: 17478510

Non-protein-coding RNAs (ncRNAs) fulfill a wide range of cellular functions from protein synthesis to regulation of gene expression. Identification of novel regulatory ncRNAs by experimental approaches commonly includes the generation of specialized cDNA libraries encoding small ncRNA species. However, such identification is severely hampered by the presence of constitutively expressed and highly abundant ‘house-keeping’ ncRNAs, such as ribosomal RNAs, small nuclear RNAs or transfer RNAs. We have developed a novel experimental strategy, designated as subtractive hybridization of ncRNA transcripts (SHORT) to specifically select and amplify novel regulatory ncRNAs, which are only expressed at certain stages or under specific growth conditions of cells. The method is based on the selective subtractive hybridization technique, formerly applied to the detection of differentially expressed mRNAs. As a model system, we applied SHORT to Epstein–Barr virus (EBV) infected human B cells. Thereby, we identified 21 novel as well as previously reported ncRNA species to be up-regulated during virus infection. Our method will serve as a powerful tool to identify novel functional ncRNAs acting as genetic switches in the regulation of fundamental cellular processes such as development, tissue differentiation or disease.

Satterfield, Brent C.; West, Jay A.A.; Caplan, Michael R.

doi: 10.1093/nar/gkm113pmid: 17517788

The majority of efforts to increase specificity or sensitivity in biosensors result in trade-offs with little to no gain in overall accuracy. This is because a biosensor cannot be more accurate than the affinity interaction it is based on. Accordingly, we have developed a new class of reagents based on mathematical principles of cooperativity to enhance the accuracy of the affinity interaction. Tentacle probes (TPs) have a hairpin structure similar to molecular beacons (MBs) for enhanced specificity, but are modified by the addition of a capture probe for increased kinetics and affinity. They produce kinetic rate constants up to 200-fold faster than MB with corresponding stem strengths. Concentration-independent specificity was observed with no false positives at up to 1 mM concentrations of variant analyte. In contrast, MBs were concentration dependent and experienced false positives above 3.88 μM of variant analyte. The fast kinetics of this label-free reagent may prove important for extraction efficiency, hence sensitivity and detection time, in microfluidic assays. The concentration-independent specificity of TPs may prove extremely useful in assays where starting concentrations and purities are unknown as would be the case in bioterror or clinical point of care diagnostics.

Grant, Gian Paola G.; Qin, Peter Z.

doi: 10.1093/nar/gkm240pmid: 17517787

In site-directed spin labeling (SDSL), a nitroxide moiety containing a stable, unpaired electron is covalently attached to a specific site within a macromolecule, and structural and dynamic information at the labeling site is obtained via electron paramagnetic resonance (EPR) spectroscopy. Successful SDSL requires efficient site-specific incorporation of nitroxides. Work reported here presents a new method for facile nitroxide labeling at the 5′ terminus of nucleic acids of arbitrary sizes. T4-polynucleotide kinase was used to enzymatically substitute a phosphorothioate group at the 5′ terminus of a nucleic acid, and the resulting phosphorothioate was then reacted with an iodomethyl derivative of a nitroxide. The method was successfully demonstrated on both chemically synthesized and naturally occurring nucleic acids. The attached nitroxides reported duplex formation as well as tertiary folding of nucleic acids, indicating that they serve as a valid probe in nucleic acid studies.

Ahlgren-Berg, Alexandra; Henriksson-Peltola, Petri; Sehlén, Wilhelmina; Haggård-Ljungquist, Elisabeth

doi: 10.1093/nar/gkm171pmid: 17485481

Bacteriophages P2 and WΦ are heteroimmune members of the P2-like family of temperate Escherichia coli phages. Temperate phages can grow lytically or form lysogeny after infection. A transcriptional switch that contains two con-vergent promoters, Pe and Pc, and two repressors regulate what life mode to enter. The immunity repressor C is the first gene of the lysogenic operon, and it blocks the early Pe promoter. In this work, some characteristics of the C proteins of P2 and WΦ are compared. An in vivo genetic analysis shows that WΦ C, like P2 C, has a strong dimerization activity in the absence of its DNA target. Both C proteins recognize two directly repeated sequences, termed half-sites and a strong bending is induced in the respective DNA target upon binding. P2 C is unable to bind to one half-site as opposed to WΦ, but both half-sites are required for repression of WΦ Pe. A reduction from three to two helical turns between the centers of the half-sites in WΦ has no significant effect on the capacity to repress Pe. However, the protein–DNA complexes formed differ, as determined by electrophoretic mobility shift experiments. A difference in spontaneous phage production is observed in isogenic lysogens.

Henriksson-Peltola, Petri; Sehlén, Wilhelmina; Haggård-Ljungquist, Elisabeth

doi: 10.1093/nar/gkm172pmid: 17412705

Bacteriophages P2, P2 Hy dis and WΦ are very similar but heteroimmune Escherichia coli phages. The structural genes show over 96% identity, but the repressors show between 43 and 63% identities. Furthermore, the operators, which contain two directly repeated sequences, vary in sequence, length, location relative to the promoter and spacing between the direct repeats. We have compared the in vivo effects of the wild type and mutated operators on gene expression with the complexes formed between the repressors and their wild type or mutated operators using electrophoretic mobility shift assay (EMSA), and real-time kinetics of the protein–DNA interactions using surface plasmon resonance (SPR) analysis. Using EMSA, the repressors formed different protein–DNA complexes, and only WΦ was significantly affected by point mutations. However, SPR analysis showed a reduced association rate constant and an increased dissociation rate constant for P2 and WΦ operator mutants. The association rate constants of P2 Hy dis was too fast to be determined. The P2 Hy dis dissociation response curves were shown to be triphasic, while both P2 and WΦ C were biphasic. Thus, the kinetics of complex formation and the nature of the complexes formed differ extensively between these very closely related phages.