Signal amplification of padlock probes by rolling circle replication.

Circularizing oligonucleotide probes (padlock probes) have the potential to detect sets of gene sequences with high specificity and excellent selectivity for sequence variants, but sensitivity of detection has been limiting. By using a rolling circle replication (RCR) mechanism, circularized but not unreacted probes can yield a powerful signal amplification. We demonstrate here that in order for the reaction to proceed efficiently, the probes must be released from the topological link that forms with target molecules upon hybridization and ligation. If the target strand has a nearby free 3' end, then the probe-target hybrids can be displaced by the polymerase used for replication. The displaced probe can then slip off the targetstrand and a rolling circle amplification is initiated. Alternatively, the target sequence itself can prime an RCR after its non-base paired 3' end has been removed by exonucleolytic activity. We found the Phi29 DNA polymerase to be superior to the Klenow fragment in displacing the target DNA strand, and it maintained the polymerization reaction for at least 12 h, yielding an extension product that represents several thousand-fold the length of the padlock probe.     

A microRNA detection system based on padlock probes and rolling circle amplification

The differential expression and the regulatory roles of microRNAs (miRNAs) are being studied intensively these years. Their minute size of only 19-24 nucleotides and strong sequence similarity among related species call for enhanced methods for reliable detection and quantification. Moreover, miRNA expression is generally restricted to a limited number of specific cells within an organism and therefore requires highly sensitive detection methods. Here we present a simple and reliable miRNA detection protocol based on padlock probes and rolling circle amplification. It can be performed without specialized equipment and is capable of measuring the content of specific miRNAs in a few nanograms of total RNA.     

Coupled rolling circle amplification loop-mediated amplification for rapid detection of short DNA sequences

Circularizable oligonucleotide probes can detect short DNA sequences with single-base resolution at the site of ligation and can be amplified by rolling circle amplification (RCA) using strand displacing polymerases. A secondary amplification scheme was developed that uses the loop-mediated amplification reaction concurrent with RCA to achieve rapid signal development from the starting circular molecules. This isothermal reaction was found to be significantly faster than the comparable hyperbranching amplification method and could detect 100 circular copies in less than 1 h.     

High accuracy genotyping directly from genomic DNA using a rolling circle amplification based assay

Background

Rolling circle amplification of ligated probes is a simple and sensitive means for genotyping directly from genomic DNA. SNPs and mutations are interrogated with open circle probes (OCP) that can be circularized by DNA ligase when the probe matches the genotype. An amplified detection signal is generated by exponential rolling circle amplification (ERCA) of the circularized probe. The low cost and scalability of ligation/ERCA genotyping makes it ideally suited for automated, high throughput methods.

Results

A retrospective study using human genomic DNA samples of known genotype was performed for four different clinically relevant mutations: Factor V Leiden, Factor II prothrombin, and two hemochromatosis mutations, C282Y and H63D. Greater than 99% accuracy was obtained genotyping genomic DNA samples from hundreds of different individuals. The combined process of ligation/ERCA was performed in a single tube and produced fluorescent signal directly from genomic DNA in less than an hour. In each assay, the probes for both normal and mutant alleles were combined in a single reaction. Multiple ERCA primers combined with a quenched-peptide nucleic acid (Q-PNA) fluorescent detection system greatly accellerated the appearance of signal. Probes designed with hairpin structures reduced misamplification. Genotyping accuracy was identical from either purified genomic DNA or genomic DNA generated using whole genome amplification (WGA). Fluorescent signal output was measured in real time and as an end point.

Conclusions

Combining the optimal elements for ligation/ERCA genotyping has resulted in a highly accurate single tube assay for genotyping directly from genomic DNA samples. Accuracy exceeded 99 % for four probe sets targeting clinically relevant mutations. No genotypes were called incorrectly using either genomic DNA or whole genome amplified sample.     

Use of synthetic oligodeoxyribonucleotides and primed cDNA as probes for pea (Pisum sativum L.) RNA and genomic DNA sequences

Two oligonucleotide sequences were synthesised by a solid-phase phosphotriester method. One of these sequences, A was a copy of part of a characterised cDNA clone encoding the basic subunit of legumin, a seed storage protein of Pisum sativum L. (garden pea); the other sequence B was predicted to be complementary to the 5 region of legumin mRNA on the basis of the amino acid sequence of legumin acidic subunits and most likely codon usage. Sequence A was shown to hybridise specifically to a legumin cDNA clone and to legumin mRNA. Sequence B did not hybridise specifically to legumin mRNA and was concluded not to be correctly complementary to legumin mRNA. Sequence A was used as a primer for cDNA synthesis using pea seed mRNA as a template. The cDNA so produced hybridised specifically to a legumin cDNA clone, to legumin mRNA, and to sequences encoding legumin in a restriction digest of pea genomic DNA. It is suggested that such oligonucleotide primed cDNAs may be of general value in probing eukaryotic genomic DNA.     

Characterization of Hordeum chilense chromosomes by C-banding and in situ hybridization using highly repeated DNA probes.

C-banding patterns of Hordeum chilense and of Triticum aestivum 'Chinese Spring' - H. chilense disomic addition lines were analyzed and compared with in situ hybridization patterns using a biotin-labeled highly repetitive Triticum tauschii DNA sequence, pAs1, and a wheat 18S-26S rDNA probe. All seven H. chilense chromosomes pairs and the added H. chilense chromosomes present in the addition lines were identified by their characteristic C-banding pattern. Chromosome morphology and banding patterns were similar to those of the corresponding chromosomes present in the parent H. chilense accession. A C-banded karyotype of the added H. chilense chromosomes was constructed and chromosome lengths, arm ratios, and relative length, as compared with chromosome 3B, were determined. The probe pAs1 was found to hybridize to specific areas on telomeres and interstitial sites along the chromosomes, allowing the identification of all seven pairs of the H. chilense chromosomes. Comparison of the patterns of distribution of the hybridization sites of clone pAs1 in the T. tauschii and H. chilense chromosomes was carried out by in situ hybridization on somatic metaphase chromosomes of the HchHchDD amphiploid. In situ hybridization using the 18S-26S rDNA probe confirmed that the H. chilense chromosomes 5Hch and 6Hch were carrying nucleolus organizer regions. The results are discussed on the basis of phylogenetic relationships between D and Hch genomes.     

Mapping of repeated DNA sequences in plant chromosomes by PRINS and C-PRINS

 The primed in situ DNA labelling (PRINS) procedure was optimised for the rapid physical mapping of several types of repetitive DNA sequences on the mitotic chromosomes of Vicia faba, Pisum sativum and Secale cereale. A localization of the highly repeated FokI sequence on V. faba chromosomes was achieved after a 7-min total reaction time. In addition, we report a procedure for direct cycling-PRINS (C-PRINS), a variation of PRINS which involves a sequence of thermal cycles analogous to the polymerase chain reaction. Compared to PRINS, C-PRINS was more sensitive. Further work is needed to improve the sensitivity of the reaction to allow for the reliable detection of low-copy DNA sequences. Received: 17 September 1996 / Accepted: 18 October 1996     

Organization and evolution of highly repeated satellite DNA sequences in plant chromosomes

A major component of the plant nuclear genome is constituted by different classes of repetitive DNA sequences. The structural, functional and evolutionary aspects of the satellite repetitive DNA families, and their organization in the chromosomes is reviewed. The tandem satellite DNA sequences exhibit characteristic chromosomal locations, usually at subtelomeric and centromeric regions. The repetitive DNA family(ies) may be widely distributed in a taxonomic family or a genus, or may be specific for a species, genome or even a chromosome. They may acquire large-scale variations in their sequence and copy number over an evolutionary time-scale. These features have formed the basis of extensive utilization of repetitive sequences for taxonomic and phylogenetic studies. Hybrid polyploids have especially proven to be excellent models for studying the evolution of repetitive DNA sequences. Recent studies explicitly show that some repetitive DNA families localized at the telomeres and centromeres have acquired important structural and functional significance. The repetitive elements are under different evolutionary constraints as compared to the genes. Satellite DNA families are thought to arise de novo as a consequence of molecular mechanisms such as unequal crossing over, rolling circle amplification, replication slippage and mutation that constitute "molecular drive".     

Detection of amplified DNA sequences by reverse chromosome painting using genomic tumor DNA as probe

A modification of reverse chromosome painting was carried out using genomic DNA from tumor cells as a complex probe for chromosomal in situ suppression hybridization to normal metaphase chromsome spreads. Amplified DNA sequences contained in such probes showed specific signals, revealing the normal chromosome positions from which these sequences were derived. As a model system, genomic DNAs were analyzed from three tumor cell lines with amplification units including the proto-oncogene c-myc. The smallest amplification unit was about 90 kb and was present in 16–24 copies; the largest unit was bigger than 600 kb and was present in 16–32 copies. Specific signals that co-localized with a differently labeled c-myc probe on chromosome band 8q24 were obtained with genomic DNA from each cell line. In further experiments, genomic DNA derived from primary tumor material was used in the case of a male patient with glioblastoma multiforme (GBM). Southern blot analysis using an epidermal growth factor receptor gene (EGFR) probe that maps to 7p13 indicated the amplification of sequences from this gene. Using reverse chromosome painting, signals were found both on band 7p13 and bands 12q13–q15. Notably, the signal on 12q13–q15 was consistently stronger. The weaker 7p13 signal showed co-localization with the major signal of the differently labeled EGFR probe. A minor signal of this probe was seen on 12q13, suggesting cross-hybridization to ERB3 sequences homologous to EGFR. The results indicate co-amplification of sequences from bands 12q13–q15, in addition to sequences from band 7p13. Several oncogenes map to 12q13–q15 providing candidate genes for a tumor-associated proto-oncogene amplification. Although the nature of the amplified sequences needs to be clarified, this experiment demonstrates the potential of reverse chromosome painting with genomic tumor DNA for rapidly mapping the normal chromosomal localization of the DNA from which the amplified sequences were derived. In addition, a weaker staining of chromosomes 10 and X was consistently observed indicating that these chromosomes were present in only one copy in the GBM genome. This rapid approach can be used to analyze cases where no metaphase spreads from the tumor material are available. It does not require any preknowledge of amplified sequences and can be applied to screen large numbers of tumors.     

The location of repeated DNA sequences in the chromosomes of Chironomus tentans

Polytene chromosomes of Chironomus tentans were hybridized in situ with in vivo labelled nuclear and chromosomal RNA. Nuclear RNA formed hybrids preferentially in five distinct regions considered to contain clustered, repeated DNA sequences. These are the two nucleolar organizer regions, Balbiani ring 1 and 2, and the 5 S RNA genes in region 2A of chromosome II, which together comprised almost 70% of the total number of grains over the complement. The remaining grains were diffusely distributed over the chromosomes. There was a significant difference in the distribution of grains when RNA from different chromosomes was used for hybridization. Chromosome I RNA hybridized preferentially with chromosome I, and chromosome II+III RNA preferentially with chromosome II+III. Some regions within the chromosomes hybridized significantly more chromosomal RNA than other regions. A considerable cross-hybridization of RNA from one particular type of chromosome with the other chromosomes was also found. It is concluded that repeated DNA sequences which hybridize with heterogeneous chromosomal RNA in C. tentans are widely dispersed in the genome. Some of these sequences have a delimited localization, others are dispersed, and some sequences which are transcribed in one particular chromosome are present also in the other chromosomes.     

Detection of target DNA using fluorescent cationic polymer and peptide nucleic acid probes on solid support

Background  

Nucleic acids detection using microarrays requires labelling of target nucleic acids with fluorophores or other reporter molecules prior to hybridization.     

A novel method to perform genomic walks using a combination of single strand DNA circularization and rolling circle amplification

Characterization of regions flanking a known sequence within a genome, known as genome walking, is a cornerstone technique in modern genetic analysis. In the present work we have developed a new PCR-dependent, directional genome walking protocol based on the unique circularization property of a novel DNA ligase, CircLigase. In the first step, PCR based primer extension is performed using a phosphorylated primer, designed to extend from the boundary of the known sequence, into the flanking region. This linear amplification results in the generation of single-stranded (ss) DNA, which is then circularized using CircLigase. Using the hyperbranching activity of Phi29 DNA polymerase, the circular ssDNA is then linearized by rolling circle amplification, resulting in copious amounts of double stranded concatameric DNA. Nested primers are used to amplify the flanking sequence using inverse PCR. The products are resolved on an agarose gel and the bands whose mobility change due to the nested location of the primer combination used are identified, extracted, and cloned into a plasmid vector for sequencing. Empirical proof for this concept was generated on two antimicrobial biosynthetic genes in Pseudomonas sp. LBUM300. Using the hcnB and phlD genes as starting points, ca 1 kb of flanking sequences were successfully isolated. The use of locus specific primers ensured both directionality and specificity of the walks, alleviating the generation of spurious amplicons, typically observed in randomly primed walking protocols. The presented genome walking protocol could be applied to any microbial genome and requires only 100-150 bp of prior sequence information. The proposed methodology does not entail laborious testing of restriction enzymes or adaptor ligation. This is the first report of a successful application of the novel ligase enzyme, CircLigase for genomic walking purposes.     

Distribution of UV-induced unscheduled DNA synthesis in metaphase chromosomes of Chinese hamster cells

Unscheduled DNA synthesis (USD) occurred in metaphase chromosomes of cultured Chinese hamster cells after ultraviolet light (UV) irradiation. When the chromosomes were labeled by UV-induced USD in metaphase, the number of grains was in proportion to the amount of chromosomal DNA and the grain densities were approximately equal all over the segments of chromosomes.     

The distribution of two highly repeated DNA sequences within Drosophila melanogaster chromosomes

In situ hybridization using ³H-RNA probes has been used to localize the sequences found in two satellites of density 1.705 g/cc and 1.672 g/ cc to specific sites within the chromosomal complement. A detailed analysis of the sites on the X chromosome was carried out using the scute series of inversions to relate the heterochromatic breakpoint relative to the location of the sequence on this chromosome. It has also been possible to establish the order of arrangement of 1.705 and 1.672 DNA at the heterochromaticeuchromatic junction on chromosome 3(R). A mitotic map is provided. The T_m of hybrids formed in situ showed that the hybrids were representative of the sequences being analyzed. The two satellites also were traced through a number of purification procedures to show that a covalent linkage may be likely between the 1.705 g/cc and 1.672 g/cc satellite as predicted from in situ hybridization analyses.     

Detection of Y-specific repeat sequences in normal and variant human chromosomes using in situ hybridization with biotinylated probes

In situ hybridization with a cloned human Y-specific repeat, pY3.4, derived from the 3.4-kb HaeIII repetitive sequences, is useful in identifying Yq-autosome translocations. In this study nonradioactive procedures were also employed to detect the sites of hybridization. Using a biotinylated probe and either immunofluorescence or horseradish peroxidase reaction, the chromosomes of three probands and members of their families with probable Y-autosome translocations were examined. It was found that not all such translocations can be correctly diagnosed based on conventional banding analysis. The present data indicate the importance of chromosome-specific probes in studying chromosome rearrangements in man.     

New sites of methylcytosine-rich DNA detected on metaphase chromosomes

In situ immunofluorescence detection of antibodies against 5-methylcytosine on metaphase chromosomes prepared by a new procedure allows the display of new 5-methylcytosine-rich sites as compared to previously published methods. In short-term culture lymphocytes, the immunofluorescent signals give a recurrent pattern in which four types of binding sites can be distinguished. Type I sites are the secondary constrictions and a few juxtacentromeric regions, type II sites correspond to T-bands. Both types I and II sites emit a strong fluorescence. Type III sites form an R-band pattern and emit a weaker fluorescence. Type IV sites are the short arms of acrocentrics, they emit strong but polymorphic signals. The results obtained from control experiments suggest that the pattern observed is rather the expression of an uneven distribution of 5-methylcytosine-rich sites than a consequence of the various treatments used. In a lymphoblastoid cell line known to have a reduced 5-methylcytosine content, it was possible to demonstrate a heterogeneous hypomethylation among chromosome structures, principally involving type I sites. The method opens the possibility of studying in situ on chromosomes, regional variations of methylation in pathological conditions.     

Genomic DNA fingerprinting of clinical isolates of Helicobacter pylori using short oligonucleotide probes containing repetitive sequences

The ability of oligonucleotide probes containing short repetitive sequence motifs to differentiate between isolates of Helicobacter pylori was investigated. Genomic DNA preparations from H. pylori were digested with the restriction enzyme Hind III, electrophoresed in agarose gels and transferred to nylon filters. Five separate oligonucleotide probes were tested for hybridization sequentially to fingerprint the digested DNA from a panel of 29 clinical isolates and one type strain of H. pylori , and their relative discriminatory abilities were assessed. Four probes, (GACA)₄, (GT)₈, (GTG)₅ and (GGAT)₄, were each shown to yield highly informative hybridization band profiles allowing differentiation of H. pylori isolates. The DNA fingerprints of individual isolates obtained with each probe were distinct and reproducible. Direct comparison with ribotyping revealed that oligonucleotide fingerprinting had far superior discriminatory power. Computer-assisted similarity analysis of (GGAT)₄-generated hybridization profiles of pairwise combinations of H. pylori isolates revealed that there was no correlation between ribotype and oligonucleotide fingerprint patterns. The results of this study demonstrate that oligonucleotide probes containing microsatellite sequences provide a new and powerful tool for isolate discrimination of H. pylori .     

The position of chromosomes at metaphase in human fibroblasts and their DNA synthesis behaviour

The peripheral location of H³-thymidine labelled chromosomes in spreads of colcemid blocked metaphases from human fibroblasts has been investigated in several karyotypes. Chromosomes show non-random distribution in relation to the periphery of the plate. Longer chromosomes are significantly more peripheral than smaller chromosomes. Those chromosomes which contain large amounts of late-synthesising DNA (Y, 18 and 13) are significantly more peripheral than early synthesising chromosomes of similar length (21–22, 19–20 and 14–15). These locations are compared with those from peripheral leucocytes obtained by other workers. When location and grain number of homologues of nos. 1, 2, 3, 16 and late-X are compared it was found that in late-S labelled cells, the more peripheral of each pair was significantly more heavily labelled; in early-S labelled cells the reverse was the case. Location and patterns of labelling between late-X homologues of XXXXY cells showed that peripheral location was related to more delay in DNA synthesis. These results are discussed in terms of peripheral and central interphase distribution of condensed chromatin. It is concluded that the apparent asynchrony of replication of homologues is principally the result of different degrees of condensation of the chromatin in the late-S phase at the time of synthesis, and these differences are related to the proximity to the nuclear membrane.     

Detection of rolling circle amplified DNA molecules using probe-tagged magnetic nanobeads in a portable AC susceptometer

Here, the volume-amplified magnetic nanobead detection assay (VAM-NDA) is for the first time applied for detection of rolling circle amplified (RCA) DNA molecules in a portable, commercial AC susceptometer that operates at ambient temperatures and with an analysis time of about 20 min. The performance of the assay is investigated using three different magnetic nanobead sizes: 50, 130 and 250nm. The performance of the assay using the AC susceptometer is compared to the performance achieved using a superconducting quantum interference device (SQUID). It is found that the performance of the assay is comparable in the two setups with a quantitative detection limit of ～4pM for all bead sizes under study. The findings show that the VAM-NDA holds promise for future wide-spread implementation in commercial AC susceptometer setups thus opening up for the possibility to perform magnetic bead-based DNA detection in point-of-care and outpatient settings.