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Proximity ligation assays are a group of protein detection techniques in which reagents with affinity for target proteins, typically antibodies, are coupled to short strands of DNA. DNA-modified affinity reagents are combined in assays constructed such that the coordinated binding of individual target molecules or complexes of interacting proteins by two or more of the reagents, followed by DNA ligation and/or polymerization reactions, gives rise to amplifiable DNA reporter strands. Proximity ligation assays have been shown to exhibit excellent sensitivity in single and multiplexed protein assays for individual or interacting proteins, both in solution and in situ. This unit describes procedures for developing solid-phase proximity ligation assays for soluble proteins using either real-time PCR or DNA sequencing as the readout. In addition, critical steps for assay optimization are discussed., (Copyright © 2015 John Wiley & Sons, Inc.)

The activity of proteins is typically regulated by secondary modifications and by interactions with other partners, resulting in the formation of protein complexes whose functions depend on the participating proteins. Accordingly, it is of central importance to monitor the presence of interaction complexes as well as their localization, thus providing information about the types of cells where the proteins are located and in what sub-cellular compartment these interactions occur. Several methods for visualizing protein interactions in situ have been developed during the last decade. These methods in most cases involve genetic constructs, and they have been successfully used in assays of living cell maintained in tissue culture, but they cannot easily be implemented in studies of clinical specimens. For such samples, affinity reagents like antibodies can be used to target the interacting proteins. In this review we will describe the in situ proximity ligation assays (in situ PLA), a method that is suitable for visualizing protein interactions in both tissue sections and in vitro cell lines, and we discuss research tasks when this or other method may be selected.

Proximity ligation assay (PLA) is a recently developed strategy for protein analysis in which antibody-based detection of a target protein via a DNA ligation reaction of oligonucleotides linked to the antibodies results in the formation of an amplifiable DNA strand suitable for analysis. Here we describe a faster and more cost-effective strategy to construct the antibody-based proximity ligation probes used in PLA that is based on the noncovalent interaction of biotinylated oligonucleotides with streptavidin followed by the interaction of this complex with biotinylated antibodies.

Methods are needed to study single molecules to reveal variability, interactions and mechanisms that may go undetected at the level of populations of molecules. We describe here an integrated series of reaction steps that allow individual nucleic acid molecules to be detected with excellent specificity. Oligonucleotide probes are circularized after hybridization to target sequences that have been prepared so that localized amplification reactions can be initiated from the target molecules. The process results in strong, discrete detection signals anchored to the target molecules. We use the method to observe the distribution, within and among human cells, of individual normal and mutant mitochondrial genomes that differ at a single nucleotide position.

Conventional cytogenetic techniques can distinguish homologous chromosomes in a qualitative manner based upon obvious morphological features or using in situ hybridization methods that yield qualitative data. We have developed a method for quantitative genotyping of single-nucleotide variants in situ using circularizable DNA probes, so-called padlock probes, targeting two different alpha satellite repeat variants present in human chromosome 7 centromeres, and a single-nucleotide variation in alpha satellite repeats on human chromosome 15 centromeres. By using these PCR-generated padlock probes, we could quantitatively distinguish homologous chromosomes and follow the transmission of the chromosomes by in situ analysis during three consecutive generations.

Highly specific and sensitive procedures will be required to evaluate proteomes. Proximity ligation is a recently introduced mechanism for protein analysis. In this technique, the convergence of sets of protein-binding reagents on individual target molecules juxtaposes attached nucleic acid sequences. Through a ligation reaction a DNA reporter sequence is created, which can be amplified. The procedure thus encodes detected proteins as specific nucleic acid sequences in what may be viewed as a reverse translation reaction.

Padlock probes are molecular tools that combine highly specific target sequence recognition with the potential for multiplexed analysis of large sets of target DNA or RNA sequences. In this brief review, we exemplify the ability of these probes to distinguish single-nucleotide target sequence variants. We further discuss means to detect the location of target sequences in situ, and to amplify reacted padlock probes via rolling-circle replication, as well as to sort reaction products on tag-arrays. We argue that the probes have the potential to render high-throughput genetic analyses precise and affordable., (Copyright 2002 Wiley-Liss, Inc.)

Oligonucleotide microarrays are used to analyze target sequences on the basis of differences in hybridization stability between matched and mismatched probe-target duplexes. DNA microarray manufacture via photolithographic synthesis generates a minority of full-length oligonucleotide probes along with a series of 5'-truncated contaminants. In a model experiment, we now investigate the effect of truncated oligonucleotides on the ability to distinguish target sequence variants that differ in a single nucleotide position. A series of oligonucleotides, mixed in proportions simulating stepwise synthetic yields of between 82 and 100%, were bound to a solid support and allowed to hybridize to a target molecule. The extent of hybridization was monitored over a range of temperatures via the fluorescence of a double-strand-specific dye. The discriminatory power of pure oligonucleotide probes was found to be significantly greater than that of a population of truncated probes, but only over a limited temperature interval. We conclude that at optimal temperatures greater oligonucleotide quality can improve the performance of oligonucleotide hybridization microarrays.

Standard fluorescence in situ hybridization (FISH) techniques using cloned probes are limited in their ability to distinguish between closely similar DNA sequences because long hybridization probes are not detectably destabilized by single mismatched base pairs. This problem has been addressed by using short allele-specific oligonucleotide probes whose hybridization to target sequences is more sensitive to mismatches. This revised and expanded unit presents protocols for discrimination between closely similar DNA sequences in situ. The discussion of probe synthesis has been greatly expanded and an Alternate Protocol 1 added for enzymatic probe ligation at low probe concentration. A new Support Protocol describes enzymatic probe synthesis.

Chromosome centromeres, composed of repeated DNA sequences, orchestrate the correct segregation of chromatids in cell division. We have examined the centromeres of human chromosomes 13 and 21 by studying the distribution, in situ, of two alpha satellite sequences that differ in a single nucleotide position. This was possible using padlock probes, oligo-nucleotides that can be ligated into circles upon target recognition. The segregation of individual 13 and 21 homologues in a family was followed by monitoring of the signals from two differentially labelled probes, specific for either sequence variant. A characteristic arrangement of the repeat motifs in three separate spots, oriented transverse to the length axis of the metaphase chromosomes and bilaterally symmetric, indicates that only parts of the detected regions are involved in the centromeric region, joining the sister chromatids before anaphase.