Glycosylases and AP-cleaving enzymes as a general tool for probe-directed cleavage of ssDNA targets.

The current arsenal of molecular tools for site-directed cleavage of single-stranded DNA (ssDNA) is limited. Here, we describe a method for targeted DNA cleavage that requires only the presence of an A nucleotide at the target position. The procedure involves hybridization of a complementary oligonucleotide probe to the target sequence. The probe is designed to create a deliberate G:A mismatch at the desired position of cleavage. The DNA repair enzyme MutY glycosylase recognizes the mismatch structure and selectively removes the mispaired A from the duplex to create an abasic site in the target strand. Addition of an AP-endonuclease, such as Endonuclease IV, subsequently cleaves the backbone dividing the DNA strand into two fragments. With an appropriate choice of an AP-cleaving enzyme, the 3'- and 5'-ends of the cleaved DNA are suitable to take part in subsequent enzymatic reactions such as priming for polymerization or joining by DNA ligation. We define suitable standard reaction conditions for glycosylase/AP-cleaving enzyme (G/AP) cleavage, and demonstrate the use of the method in an improved scheme for in situ detection using target-primed rolling-circle amplification of padlock probes.

A single molecule array for digital targeted molecular analyses.

We present a new random array format together with a decoding scheme for targeted multiplex digital molecular analyses. DNA samples are analyzed using multiplex sets of padlock or selector probes that create circular DNA molecules upon target recognition. The circularized DNA molecules are amplified through rolling-circle amplification (RCA) to generate amplified single molecules (ASMs). A random array is generated by immobilizing all ASMs on a microscopy glass slide. The ASMs are identified and counted through serial hybridizations of small sets of tag probes, according to a combinatorial decoding scheme. We show that random array format permits at least 10 iterations of hybridization, imaging and dehybridization, a process required for the combinatorial decoding scheme. We further investigated the quantitative dynamic range and precision of the random array format. Finally, as a demonstration, the decoding scheme was applied for multiplex quantitative analysis of genomic loci in samples having verified copy-number variations. Of 31 analyzed loci, all but one were correctly identified and responded according to the known copy-number variations. The decoding strategy is generic in that the target can be any biomolecule which has been encoded into a DNA circle via a molecular probing reaction.

Proximity ligation assays: a recent addition to the proteomics toolbox.

An essential skill for every researcher is to learn how to select and apply the most appropriate methods for the questions they are trying to answer. With the extensive variety of methods available, it is increasingly important to scrutinize the advantages and disadvantages of these techniques prior to making a decision on which to use. In this article, we describe an approach to evaluate methods by reducing them into subcomponents. This is exemplified by a brief description of some commonly used proteomics methods. The same approach can also be used in method development by rearranging subcomponents in order to create new methods, as demonstrated with the development of proximity ligation assays (PLAs). PLA is a method as designed in our laboratory for detection of proteins, protein-protein interactions and post-translational modifications. Fundamentally, protein-recognition events are converted into detectable DNA molecules. The technique uses protein-DNA conjugates as binders for the targets of interest. Binding of two or more conjugates to the target results in assembly of an assay-specific DNA molecule. Subsequent amplification of the DNA molecule generates a signal that can be detected using PCR, for detection of minute amounts of proteins in serum, or standard fluorescence microscopy for detection of protein-protein interactions in tissue sections. Lastly, we apply the approach of recombining subcomponents to develop a few novel hypothetical methods hoping this might stimulate the readers to utilize this approach themselves.

Detection of DNA hybridization using induced fluorescence resonance energy transfer.

Induced fluorescence resonance energy transfer (iFRET) is a variation of resonance energy transfer that is particularly well-suited for the detection of DNA hybridization. The underlying mechanism involves monitoring changes in fluorescence that are the result of an energy transfer reaction between a specific pair of donor and acceptor moieties. In iFRET, the donor is a dye that only fluoresces while interacting with double-stranded DNA and the acceptor is dye that is covalently linked to an oligonucleotide probe. Hybridization of the probe to its complement induces excitement of the donor dye and subsequent energy transfer to the acceptor dye. The energy transfer reaction (and concomitant hybridization status) can easily be followed by monitoring the fluorescence output of the acceptor dye. Because the interaction of the donor dye is reversible and dependent on the presence of double-stranded DNA, iFRET is extremely useful and herein demonstrated in the generation of DNA melting curves.

Creating arrays by centrifugation.

We describe afast, low-cost, and reliable way of creating arrays from sample molecules of interest present within microformatted sample vessels (such as 1536-well microplates). The principle involves simple centrifugal transfer of molecules of interest onto a solid planar or membrane surfaces placed over the initial sample vessel. Tools and procedures are presented that validate the robustness and precision of this facile solution to an otherwise difficult problem in modern molecular genetics. The availability of transferred DNA molecules for hybridization is also demonstrated. In conclusion, this "centrifugal-array" concept should help research studies to be applied on ever-greater scales with very simple machinery.

Scoring insertion-deletion polymorphisms by dynamic allele-specific hybridization.

Genome variation provides researchers with thousands of markers with which to study human demographic history and phenotypes. Insertion-deletion (indel) polymorphism is an important and abundant form of human genome variation, and convenient methods for genotyping indels are therefore needed. Here we evaluate dynamic allele-specific hybridization (DASH) for its ability to score indels. Evaluation of six model indel DASH assays based on synthetic oligonucleotides showed that length differences of 1-5 bp were accurately scored. Only single probes were required to assay indels of 3-4 bp or less, while longer indels tended to require the use of both allele probes serially. The best results were obtained by central placing of the probe over the indel. Model study findings were confirmed by running indel DASH assays upon PCR-amplified targets representing four polymorphisms from Alzheimer's disease candidate genes APBB1 and LRP1. These indels were genotyped in a set of 121 patients and 156 controls. While no disease association was found, the data quality confirmed that DASH is a robust and useful procedure for genotyping indels of the size range typically found in the human genome.

Evaluation of multiple presenilin 2 SNPs for association with early-onset sporadic Alzheimer disease.

The presenilin genes encode proteins that modify, mediate, or perform similar functions to gamma-secretase, the enzyme responsible for converting amyloid beta precursor protein (APP) into beta-amyloid. Mutations in the presenilin genes cause an increased production of Abeta42, the aberrant form of beta-amyloid found in the neural plaques of Alzheimer disease patients. Previously reported association studies of presenilin 2 (PSEN2) polymorphisms with early-onset Alzheimer disease (EOAD) have produced contradictory results. In an effort to resolve these differences, we tested eight single nucleotide polymorphisms in and around the 3' region of the PSEN2 gene for association with EOAD. An initial set of Scottish EOAD cases (n = 121) and controls (n = 152) was screened using the genotyping method dynamic allele-specific hybridization (DASH). No significant differences were seen between allele or genotype frequencies of cases and controls. However, when conditioned on the risk allele (epsilon 4) APOE, three polymorphisms showed allelic association with a P value below 0.05. These same polymorphisms were in near 100% linkage disequilibrium with each other (P < 5 x 10(-5)), and in each, one of the homozygous genotypes was absent in controls but present in the cases. Replication in an independent set of Scottish EOAD cases (n = 84) and controls (n = 173) did not confirm this finding. From this study we find no evidence to suggest that variations in the PSEN2 gene pose as major risk factors for sporadic EOAD.

Rapid identification of bio-molecules applied for detection of biosecurity agents using rolling circle amplification.

Detection and identification of pathogens in environmental samples for biosecurity applications are challenging due to the strict requirements on specificity, sensitivity and time. We have developed a concept for quick, specific and sensitive pathogen identification in environmental samples. Target identification is realized by padlock- and proximity probing, and reacted probes are amplified by RCA (rolling-circle amplification). The individual RCA products are labeled by fluorescence and enumerated by an instrument, developed for sensitive and rapid digital analysis. The concept is demonstrated by identification of simili biowarfare agents for bacteria (Escherichia coli and Pantoea agglomerans) and spores (Bacillus atrophaeus) released in field.

iFRET: an improved fluorescence system for DNA-melting analysis.

Fluorescence resonance energy transfer (FRET) is a powerful tool for detecting spatial relationships between macromolecules, one use of which is the tracking of DNA hybridization status. The process involves measuring changes in fluorescence as FRET donor and acceptor moieties are brought closer together or moved farther apart as a result of DNA hybridization/denaturation. In the present study, we introduce a new version of FRET, which we term induced FRET (iFRET), that is ideally suited for melting curve analysis. The innovation entails using a double-strand, DNA-specific intercalating dye (e.g., SYBR Green I) as the FRET donor, with a conventional FRET acceptor affixed to one of the DNA molecules. The SNP genotyping technique dynamic allele specific hybridization (DASH) was used as a platform to compare iFRET to two alternative fluorescence strategies, namely, the use of the intercalating dye alone and the use of a standard FRET pair (fluorescein as donor, 6-rhodamine as acceptor). The iFRET configuration combines the advantages of intercalating dyes, such as high signal strengths and low cost, with maintaining the specificity and multiplex potential afforded by traditional FRET detection systems. Consequently, iFRET represents a fresh and attractive schema for monitoring interactions between DNA molecules.

DASH-2: flexible, low-cost, and high-throughput SNP genotyping by dynamic allele-specific hybridization on membrane arrays.

Genotyping technologies need to be continually improved in terms of their flexibility, cost-efficiency, and throughput, to push forward genome variation analysis. To this end, we have leveraged the inherent simplicity of dynamic allele-specific hybridization (DASH) and coupled it to recent innovations of centrifugal arrays and iFRET. We have thereby created a new genotyping platform we term DASH-2, which we demonstrate and evaluate in this report. The system is highly flexible in many ways (any plate format, PCR multiplexing, serial and parallel array processing, spectral-multiplexing of hybridization probes), thus supporting a wide range of application scales and objectives. Precision is demonstrated to be in the range 99.8-100%, and assay costs are 0.05 USD or less per genotype assignment. DASH-2 thus provides a powerful new alternative for genotyping practice, which can be used without the need for expensive robotics support.