Search

Multiplex protein quantification has been constrained by issues of assay specificity, sensitivity and throughput. This research presents a novel approach that overcomes these limitations using antibody-oligonucleotide conjugates for immuno-polymerase chain reaction (immuno-PCR) or proximity ligation, coupled with competitive PCR and MALDI-TOF mass spectrometry. Employing these combinations of technologies, we demonstrate multiplex detection and quantification of up to eight proteins, spanning wide dynamic ranges from femtomolar concentrations, using only microliter sample volumes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Gene expression - a key feature for modulating cell fate-is regulated in part by histone modifications, which modulate accessibility of the chromatin to transcription factors. Until now, protein-DNA interactions (PDIs) have mostly been studied in bulk without retrieving spatial information from the sample or with poor sequence resolution. New tools are needed to reveal proteins interacting with specific DNA sequences in situ for further understanding of the orchestration of transcriptional control within the nucleus. We present herein an approach to visualise individual PDIs within cells, based on the in situ proximity ligation assay (PLA). This assay, previously used for the detection of protein-protein interactions in situ, was adapted for analysis of target PDIs, using padlock probes to identify unique DNA sequences in complex genomes. As a proof-of-principle we detected histone H3 interacting with a 26 bp consensus sequence of the Alu-repeat abundantly expressed in the human genome, but absent in mice. However, the mouse genome contains a highly similar sequence, providing a model system to analyse the selectivity of the developed methods. Although efficiency of detection currently is limiting, we conclude that in situ PLA can be used to achieve a highly selective analysis of PDIs in single cells., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Improved protein assays promise to offer new insights into biological processes as well as the identification of new, clinically important biomarkers. In recent years, a number of approaches have been developed where protein-binding reagents, typically antibodies, are equipped with DNA strands to enable protein analyses via powerful nucleic acid detection reactions for improved performance. In this review, we provide a background to this emerging field, and we describe several different ways in which these reagents can improve protein analyses by lowering detection thresholds, improving multiplexing and extending the range of biomolecules available for analysis, both in research settings and in clinical routine., (© 2012 Expert Reviews Ltd)

Analytical reactions in which short DNA strands are used in combination with DNA ligases have proven useful for measuring, decoding, and locating most classes of macromolecules. Given the need to accumulate large amounts of precise molecular information from biological systems in research and in diagnostics, ligation reactions will continue to offer valuable strategies for advanced analytical reactions. Here, we provide a basis for further development of methods by reviewing the history of analytical ligation reactions, discussing the properties of ligation reactions that render them suitable for engineering novel assays, describing a wide range of successful ligase-based assays, and briefly considering future directions.

Protein-binding DNA sequence elements encode a variety of regulated functions of genomes. Information about such elements is currently in a state of rapid growth, but improved methods are required to characterize the sequence specificity of DNA-binding proteins. We have established an in vitro method for specific and sensitive solution-phase analysis of interactions between proteins and nucleic acids in nuclear extracts, based on the proximity ligation assay. The reagent consumption is very low, and the excellent sensitivity of the assay enables analysis of as few as 1-10 cells. We show that our results are highly reproducible, quantitative, and in good agreement with both EMSA and predictions obtained by using a motif finding software. This assay can be a valuable tool to characterize in-depth the sequence specificity of DNA-binding proteins and to evaluate effects of polymorphisms in known transcription factor binding sites.

We present a tightly controlled process for strand-specific amplification of circularized DNA molecules. Tandem repeated complements of DNA circles are generated by rolling-circle replication, and converted to monomer circles of opposite polarity to that of the starting material. These circles are then subjected to one more round of rolling-circle replication and circularization, and the process can be further repeated. The method can be directed to produce single-stranded circular or linear monomers, or linear concatemers of the desired polarity. The reaction is not product inhibited, and can yield approximately 100-fold higher concentrations of monomer products than PCR. Each generation of the amplification process proceeds in a linear fashion, ensuring precise quantification. The procedure is suitable for parallel amplification of large numbers of DNA circles, because the few cycles and the robust reaction mechanism preserves the proportion of amplified molecules. We demonstrate the utility of the method for multiplexed genotyping of polymorphic loci and for quantitative DNA analysis.

Branched nucleic acid strands exist as intermediates in certain biological reactions, and bifurcating DNA also presents interesting opportunities in biotechnological applications. We describe here how T4 DNA ligase can be used for efficient construction of DNA molecules having one 5' end but two distinct 3' ends that extend from the 2' and 3' carbons, respectively, of an internal nucleotide. The nature of the reaction products is investigated, and optimal reaction conditions are reported for the construction of branched oligonucleotides. We discuss the utility of these branched DNA nanostructures for gene detection.

The advent of in vitro DNA amplification has enabled rapid acquisition of genomic information. We present here an analogous technique for protein detection, in which the coordinated and proximal binding of a target protein by two DNA aptamers promotes ligation of oligonucleotides linked to each aptamer affinity probe. The ligation of two such proximity probes gives rise to an amplifiable DNA sequence that reflects the identity and amount of the target protein. This proximity ligation assay detects zeptomole (40 x 10(-21) mol) amounts of the cytokine platelet-derived growth factor (PDGF) without washes or separations, and the mechanism can be generalized to other forms of protein analysis.

Ligase-mediated gene detection has proven valuable for detection and precise distinction of DNA sequence variants. We have recently shown that T4 DNA ligase can also be used to distinguish single nucleotide variants of RNA sequences. Here we describe parameters that influence RNA-templated DNA ligation by T4 DNA ligase. The reaction proceeds much more slowly, requiring more enzyme, compared to ligation of the same oligonucleotides hybridized to the corresponding DNA sequence. The reaction is inhibited at high concentrations of ATP and NaCl and both magnesium and manganese ions can support the reaction. We define reaction conditions where 80% of RNA target molecules can template a diagnostic ligation reaction. Ligase-mediated RNA detection should provide a useful mechanism for sensitive and accurate detection and distinction of RNA sequence variants.

Nucleotide sequence information derived from DNA segments of the human and other genomes is accumulating rapidly. However, it frequently proves difficult to use such short DNA segments to identify clones in genomic libraries or fragments in blots of the whole genome or for in situ analysis of chromosomes. Oligonucleotide probes, consisting of two target-complementary segments, connected by a linker sequence, were designed. Upon recognition of the specific nucleic acid molecule the ends of the probes were joined through the action of a ligase, creating circular DNA molecules catenated to the target sequence. These probes thus provide highly specific detection with minimal background.

Several techniques exist that permit the efficient distinction among characterized DNA sequence variants. In this review we discuss a number of such analytic procedures. These techniques all take advantage of a variety solid supports to prepare and analyze reaction products. The described diagnostic principles are now being applied for the development of miniaturized assay formats, suitable for automated detection of large sets of sequences in clinical samples.

A number of novel gene detection techniques all revolve around the ligation of synthetic nucleic acid probes. In such ligase-assisted gene detection reactions, specific DNA or RNA sequences are investigated by using them as guides for the covalent joining of pairs of probe molecules. The probes are designed to hybridize immediately next to each other on the target nucleic acid strand. Demonstration of ligated probes results in highly specific detection of and efficient distinction between similar sequence variants under standard reaction conditions. Accordingly, the principle has been applied in automated genetic screening procedures. Ligation reactions are also integral to a number of amplification procedures and they will be of value in an expanding range of genetic analyses.

Protocols for in vitro amplification of nucleic acids are proliferating and there are now several methods that will contribute both to genetic research and to the diagnosis of a wide range of diseases. Here, I present the working principles of some of these molecular machines for amplifying DNA or RNA and discuss the lines along which new methods of amplifying nucleic acids may be developed.

We have devised a procedure, termed capture PCR (CPCR), that permits the rapid isolation of DNA segments situated adjacent to a characterized nucleotide sequence. In this procedure, a DNA sample is restriction-digested and a linker, comprising two base-paired oligonucleotides, is added to the ends by ligation. Multiple extension reactions are performed using a biotinylated primer derived from the known sequence, permitting the subsequent isolation of extension products on a streptavidin-coated support. The enriched fragments are amplified exponentially using another specific oligonucleotide, hybridizing 3' to the biotinylated primer in combination with one of the linker oligonucleotides, now functioning as a PCR primer. The convenience of CPCR is greatly enhanced by using a novel streptavidin-coated manifold, which is constructed so that it projects into each individual well of a microtiter plate. The procedure permits the simultaneous isolation of fragments from large numbers of DNA samples and minimizes the risk of contamination between reactions. We have applied this method to identify DNA sequences located downstream of known sequences in the human genome. The technique has also been used to identify end fragments of sequences cloned in a yeast artificial chromosome (YAC) vector. The reactions can be initiated directly from yeast colonies and provide access to DNA sequence information for these end fragments in a minimal number of steps. With the aid of the present technique, we have isolated over 100 end fragments from YACs derived from the human X chromosome. Isolated end sequences have been used to order YAC clones into a contig.

Methodological improvements in molecular genetics will provide inexpensive, efficient methods suitable for routine clinical diagnosis in a wide range of conditions. Assay sensitivity is greatly enhanced by pre-amplification of the target nucleic acid segment, enabling extremely scant tissue samples to be analysed and low grade infections to be detected. Reagent kits and instruments are becoming available for detecting specific nucleic acids, distinguishing variant sequences, and establishing copy number; and RNA analysis will furnish insights into a diversity of acute reactions. However, perhaps the most important benefit of DNA diagnostics will be to open up predictives perspectives in medicine.

DNA diagnostics, the detection of specific DNA sequences, will play an increasingly important role in medicine as the molecular basis of human disease is defined. Here, we demonstrate an automated, nonisotopic strategy for DNA diagnostics using amplification of target DNA segments by the polymerase chain reaction (PCR) and the discrimination of allelic sequence variants by a colorimetric oligonucleotide ligation assay (OLA). We have applied the automated PCR/OLA procedure to diagnosis of common genetic diseases, such as sickle cell anemia and cystic fibrosis (delta F508 mutation), and to genetic linkage mapping of gene segments in the human T-cell receptor beta-chain locus. The automated PCR/OLA strategy provides a rapid system for diagnosis of genetic, malignant, and infectious diseases as well as a powerful approach to genetic linkage mapping of chromosomes and forensic DNA typing.

Molecular biology has revolutionized the understanding of many aspects of human disease. Ongoing developments in DNA diagnostics--the analysis of disease at the nucleic acid level--will soon provide automated, rapid, and inexpensive analyses for DNA or RNA sequences associated with genetic, malignant, and infectious diseases. DNA diagnostics will also facilitate the identification of disease-associated genes at birth, thus creating new opportunities for preventive medicine.

A two-step strategy is described here to rapidly analyze gene-sequence variation or polymorphism. First, DNA sequences flanking the coding region of the gene to be analyzed are determined directly from a cosmid clone, including the gene, using the modified T7 DNA polymerase and sequencing primers based on the cDNA sequence of the gene. Second, the identified gene-flanking sequences are used to design amplification primers for the polymerase chain reaction (PCR) to permit amplification of DNA segments of up to 1 kilobase in genomic DNA from multiple individuals. These amplified DNA segments are directly sequenced using the thermostable Taq DNA polymerase.

An assay for the presence of given DNA sequences has been developed, based on the ability of two oligonucleotides to anneal immediately adjacent to each other on a complementary target DNA molecule. The two oligonucleotides are then joined covalently by the action of a DNA ligase, provided that the nucleotides at the junction are correctly base-paired. Thus single nucleotide substitutions can be distinguished. This strategy permits the rapid and standardized identification of single-copy gene sequences in genomic DNA.