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Recent outbreaks of avian influenza in different parts of the world have caused major economic losses for the poultry industry, affected wildlife seriously and present a significant threat even to human public health, due to the risk for zoonotic transmission. The ability to recognize avian influenza viruses (AIVs) early is of paramount importance to ensure that appropriate measures can be taken quickly to contain the outbreak. In this study, the performance of a proximity ligation assay (PLA) for the detection of AIV antigens in biological specimens was evaluated. It is shown that PLA: (i) as a novel principle of highly sensitive antigen detection is extending the arsenal of tools for the diagnosis of AIV; (ii) is very specific, nearly as sensitive as a commonly used reference real-time PCR assay, and four orders of magnitude more sensitive than a sandwich ELISA, utilizing the same antibody; (iii) avoids the necessity of nucleic acids extraction, which greatly facilitates high-throughput implementations; (iv) allows the use of inactivated samples, which safely can be transported from the field to diagnostic laboratories for further analysis. In summary, the results demonstrate that PLA is suited for rapid, accurate and early detection of AIV.

A new concept for multiplex detection and quantification of microbes is here demonstrated on a range of infectious fungal species. Padlock probe methodology in conjunction with qPCR and Luminex technology was used for simultaneous detection of ten fungal species in one single experiment. By combining the multiplexing properties of padlock probes and Luminex detection with the well established quantitative characteristics of qPCR, quantitative microbe detection was done in 10-plex mode. A padlock probe is an oligonucleotide that via a ligation reaction forms circular DNA when hybridizing to specific target DNA. The region of the padlock probe that does not participate in target DNA hybridization contains generic primer sequences for amplification and a tag sequence for Luminex detection. This was the fundament for well performing multiplexing. Circularized padlock probes were initially amplified by rolling circle amplification (RCA), followed by a SybrGreen real time PCR which allowed an additive quantitative assessment of target DNA in the sample. Detection and quantification of amplified padlock probes were then done on color coded Luminex microspheres carrying anti-tag sequences. A novel technique, using labeled oligonucleotides to prevent reannealing of amplimers by covering the flanks of the address sequence, improved the signal to noise ratio in the detection step considerably. The method correctly detected fungi in a variety of clinical samples and offered quantitative information on fungal nucleic acid.

We present a method to specifically select large sets of DNA sequences for parallel amplification by PCR using target-specific oligonucleotide constructs, so-called selectors. The selectors are oligonucleotide duplexes with single-stranded target-complementary end-sequences that are linked by a general sequence motif. In the selection process, a pool of selectors is combined with denatured restriction digested DNA. Each selector hybridizes to its respective target, forming individual circular complexes that are covalently closed by enzymatic ligation. Non-circularized fragments are removed by exonucleolysis, enriching for the selected fragments. The general sequence that is introduced into the circularized fragments allows them to be amplified in parallel using a universal primer pair. The procedure avoids amplification artifacts associated with conventional multiplex PCR where two primers are used for each target, thereby reducing the number of amplification reactions needed for investigating large sets of DNA sequences. We demonstrate the specificity, reproducibility and flexibility of this process by performing a 96-plex amplification of an arbitrary set of specific DNA sequences, followed by hybridization to a cDNA microarray. Eighty-nine percent of the selectors generated PCR products that hybridized to the expected positions on the array, while little or no amplification artifacts were observed.

We describe PieceMaker, a software tool for the design of applications of selector probes-oligonucleotide probes that direct circularization of target nucleic acid molecules. Such probes can be combined in parallel to circularize a selection of fragments from restriction digested total genomic DNA. These fragments can then be amplified in a single PCR using a common primer pair, yielding substrates for subsequent analyses, such as parallel genotyping or sequencing. However, designing multiplex selector assays is a laborious task. The PieceMaker program alleviates this problem by selecting restriction enzymes to generate suitable fragments for selection, and generating the output data required to design the selector probes.

We report on the development of molecular inversion probe (MIP) genotyping, an efficient technology for large-scale single nucleotide polymorphism (SNP) analysis. This technique uses MIPs to produce inverted sequences, which undergo a unimolecular rearrangement and are then amplified by PCR using common primers and analyzed using universal sequence tag DNA microarrays, resulting in highly specific genotyping. With this technology, multiplex analysis of more than 1,000 probes in a single tube can be done using standard laboratory equipment. Genotypes are generated with a high call rate (95%) and high accuracy (>99%) as determined by independent sequencing.

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.

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.)

There is increasing demand for efficient methods to relate genomic information from model organisms to other species of interest. Comparative genetic analyses are particularly valuable to identify functionally important sequence features on the basis of their evolutionary conservation. We demonstrate here how a single segment of just 32 or less conserved coding nucleotide positions can be used to isolate homologous gene sequences from large numbers of species using a single-sided PCR technique. The method was used to isolate and determine the 3'-untranslated sequence of the somatostatin gene from vertebrate species ranging from human to hagfish. Two sequence motifs centered an average 40-145 nucleotides downstream of the translational stop codon have remained conserved for up to 350 million years. One of the conserved tetrapod segments was used to select a primer for amplification of so-called comparative anchor tagged sequences (CATS) in regular PCR, and shown to amplify homologous sequences from DNA samples from 30 out of 33 tetrapods. In conclusion, we present a useful procedure to reveal functionally relevant sequence elements, and to select primers for amplification of homologous sequences from a wide range of species.

Circularizing oligonucleotide probes, so-called padlock probes, have properties that should prove valuable in a wide range of genetic investigations, including in situ analyses, genotyping and measurement of gene expression. However, padlock probes can be difficult to obtain by standard oligonucleotide synthesis because they are relatively long and require intact 5'- and 3'-end sequences to function. We describe a PCR-based protocol for flexible small-scale enzymatic synthesis of such probes. The protocol also offers the advantage over chemical synthesis that longer probes can be made that are densely labeled with detectable functions, resulting in an increased detection signal. The utility of probes synthesized according to this protocol is demonstrated for the analysis of single nucleotide variations in human genomic DNA both in situ and in solution.

A modified capture polymerase chain reaction (CPCR) technique was used to isolate the entire sequence of the nifH gene and its flanking regions from a natural population of Trichodesmium sp. A set of specific CPCR primers derived from a known 72-bp DNA segment of the nifH sequence permitted isolation of both the upstream and the downstream region of Trichodesmium sp. nifH. The 882-bp nifH gene presented here is the first full-length gene isolated from Trichodesmium sp. A sequence similar to a nif-like promoter was found in front of nifH. The nifH open reading frame of Trichodesmium sp. encoded 294 amino acids. Comparative analysis of the Trichodesmium sp. NifH sequence revealed strong similarity with 23 known NifH proteins. Amino acids postulated to be involved in binding of the 4Fe:4S cluster and those subjected to ADP-ribosylation were present. An open reading frame for the nifD gene was identified 189 bp downstream of nifH. A sequence similar to the consensus of the nif-like promoter was also found in front of nifD.

A mutation that disrupts the gene for one of the major proteins in tooth enamel has been investigated. The mutation is located in the amelogenin gene and causes X-linked amelogenesis imperfecta, characterized by defective mineralization of tooth enamel. We have isolated the breakpoints of a 5-kb deletion in the amelogenin gene on the basis of nucleotide sequence information located upstream of the lesion, using a technique termed capture PCR. The deletion removes five of the seven exons, spanning from the second intron to the last exon. Only the first two codons for the mature protein remain, consistent with the relatively severe phenotype of affected individuals in the present family. The mutation appears to have arisen as an illegitimate recombination event since of 11 nucleotide positions immediately surrounding the two breakpoints, 9 are identical.

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.

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.