DNA Rolling Circle Amplification Research Articles

Nanoplasmonic Rapid Antimicrobial-Resistance Point-of-Care Identification Device: RAPIDx.

The emergence of antibiotic resistance has become a global health crisis, and everyone must arm themselves with wisdom to effectively combat the "silent tsunami" of infections that are no longer treatable with antibiotics. However, the overuse or inappropriate use of unnecessary antibiotics is still routine for administering them due to the unavailability of rapid, precise, and point-of-care assays. Here, a rapid antimicrobial-resistance point-of-care identification device (RAPIDx) is reported for the accurate and simultaneous identification of bacterial species (genotype) and target enzyme activity (phenotype). First, a contamination-free active target enzyme is extracted via the photothermal lysis of preconcentrated bacteria cells on a nanoplasmonic functional layer on-chip. Second, the rapid, precise identification of pathogens is achieved by the photonic rolling circle amplification of DNA on a chip. Third, the simultaneous identification of bacterial species (genotype) and target enzyme activity (phenotype) is demonstrated within a sample-to-answer 45min operation via the RAPIDx. It is believed that the RAPIDx will be a valuable method for solving the bottleneck of employing on-chip nanotechnology for antibiotic-resistant bioassay and other infectious diseases.

Diversity and phylogeography of begomoviruses and DNA satellites associated with the leaf curl and mosaic disease complex of eggplant

Eggplant is one of the important vegetable crops grown across the world, and its production is threatened by both biotic and abiotic stresses. Diseases caused by viruses are becoming major limiting factors for its successful cultivation. A survey for begomovirus-like symptoms in 72 eggplant fields located in six different Indian states revealed a prevalence of disease ranging from 5.2 to 40.2%, and the symptoms recorded were mosaic, mottling, petiole bending, yellowing, and upward curling, vein thickening, and enation of the leaves, and stunting of plants. The causal agent associated with these plants was transmitted from infected leaf samples to healthy eggplant seedlings via grafting and whiteflies (Bemisia tabaci). The presence of begomovirus was confirmed in 72 infected eggplant samples collected from the surveyed fields exhibiting leaf curl and mosaic disease by PCR using begomovirus specifc primers (DNA-A componet), which resulted in an expected amplicon of 1.2 kb. The partial genome sequence obtained from amplified 1.2 kb from all samples indicated that they are closely related begomovirus species, tomato leaf Karnataka virus (ToLCKV, two samples), tomato leaf curl Palampur virus (ToLCPalV, fifty eggplant samples), and chilli leaf curl virus (ChLCuV, twenty samples). Based on the partial genome sequence analysis, fourteen representative samples were selected for full viral genome amplification by the rolling circle DNA amplification (RCA) technique. Analyses of fourteen eggplant isolates genome sequences using the Sequence Demarcation Tool (SDT) indicated that one isolate had the maximum nucleotide (nt) identity with ToLCKV and eight isolates with ToLCPalV. Whereas, four isolates four isolates (BLC1-CH, BLC2-CH, BLC3-CH, BLC4-CH) are showing nucleotide identity of less than 91% with chilli infecting viruses begomoviruses with chilli infecting begomoviruses and as per the guidelines given by the ICTV study group for the classification of begomoviruses these isolates are considered as one novel begomovirus species, for which name, Eggplant leaf curl Chhattisgarh virus (EgLCuChV) is proposed. For DNA-B component, seven eggplant isolates had the highest nt identity with ToLCPalV infecting other crops. Further, DNA satellites sequence analysis indicated that four betasatellites identified shared maximum nucleotide identity with the tomato leaf curl betasatellite and five alphasatellites shared maximum nucleotide identity with the ageratum enation alphasatellite. Recombination and GC plot analyses indicated that the bulk of begomovirus genome and associated satellites presumably originated from of previously known mono and bipartite begomoviruses and DNA satellites. To the best of our knowledge, this is India's first report of ToLCKV and a noval virus, eggplant leaf curl Chhattisgarh virus associated with eggplant leaf curl disease.

Identification of Novel Begomoviruses Associated with Leaf Curl Disease of Papaya (Carica papaya L.) in India

Papaya (Carica papaya L.) is one of the most important fruit crops grown in tropical and subtropical regions of the world. Papaya leaf curl disease is one of the greatest concerns next to Papaya ring spot disease for India and the world. A survey was conducted during the year 2019 to 2021 for assessing the leaf curl disease incidence in five major papaya-growing districts of Karnataka State, India. The incidence ranged from 10 to 21 percent, with plants expressing typical begomovirus symptoms. Thirty-two virus-infected papaya samples (PLC-1 to PLC-32), collected from different farmer’s fields, gave positive amplification for begomovirus detection. Based on the partial genome analysis, 13 representative papaya leaf curl isolates were selected for complete genome amplification by rolling circle DNA amplification (RCA). The RCA products were cloned, sequenced and analyzed. Based on the analysis and strain classification criteria for begomoviruses, five isolates (PLC-2, 3, 9, 11 and 18) were considered variants of Chilli leaf curl virus (ChiLCV). Isolate PLC-22 is considered a strain of ChiLCV, with 93.5% nt identity sharing. Similarly, isolate PLC-28 is considered a strain of Croton yellow vine mosaic virus (CYVMV), and isolates PLC-25 and PLC-31 were considered as strains of Papaya leaf curl virus (PaLCuV). Among the remaining four isolates, three (PLC-1, PLC-4 and PLC-7) share more than 91% nt identity among them and less than 91% nt identity with all other reported begomovirus isolates. Hence, they are considered to be isolates of the novel begomovirus, and the name Papaya leaf curl Bagalkote virus [India:Karnataka:Bagalkote:Papaya:2021] is proposed. One isolate (PLC-32) is also found to be distinct from all other begomovirus isolates, including the isolates in the current study also considered to be novel begomovirus, for which we propose the name Papaya leaf curl Haveri virus [India:Karnataka:Haveri:Papaya:2021]. The putative recombination analysis of all 13 papaya isolates showed that a major part of the viral genome was likely descended from the begomoviruses reported previously. This is the first report on the diversity and a distribution of the begomoviruses infecting papaya in Karnataka, India. The current investigation results revealed five major papaya-infecting begomoviruses (PaLCuBKV, ChiLCV, PaLCuV, CYVMV and PaLCuHV) in the sampled regions.

Ionic Signal Enhancement by the Space Charge Effect through the DNA Rolling Circle Amplification on the Outer Surface of Nanochannels.

DNA species are recognized as a powerful probe for nanochannel analyses to address the issues of specific target recognition and highly efficient signal conversion due to their programmable and predictable Watson-Crick bases. However, in the conventional view, abundant sophisticated DNA structures synthesized by DNA amplification strategies are unsuitable for use in nanochannel analyses owing to their low probability to enter a nanochannel restricted by the smaller opening of the nanochannel, as well as the faint ion signal produced by the steric effect. Here, we present an integrated strategy of nanochannel analyses that combines the target recognitions by encoded rolling circle amplification (RCA) in solution and the ionic signal enhancement by the space charge effect through the immobilization of highly negative-charged RCA amplicons on the outer surface of the nanochannels. Owing to the highly negative-charged RCA amplicons with 100 nm sizes, a sharp increase of ionic current up to 7454% has been achieved. The RCA amplicon triggered by mRNA-21 on the outer surface of the poly(ethylene terephthalate) membrane with a single nanochannel realized the single-base mismatch detection of mRNA-21 with a sensitivity of 6 fM. The DNA amplicon endows the nanochannel with high sensitivity and selectivity that could extend to other applications, such as DNA sequencing, desalination, sieving, and water-energy nexus.

DNA flowerstructure co-localizes with human pathogens in infected macrophages.

Herein, we characterize the cellular uptake of a DNA structure generated by rolling circle DNA amplification. The structure, termed nanoflower, was fluorescently labeled by incorporation of ATTO488-dUTP allowing the intracellular localization to be followed. The nanoflower had a hydrodynamic diameter of approximately 300 nanometer and was non-toxic for all mammalian cell lines tested. It was internalized specifically by mammalian macrophages by phagocytosis within a few hours resulting in specific compartmentalization in phagolysosomes. Maximum uptake was observed after eight hours and the nanoflower remained stable in the phagolysosomes with a half-life of 12 h. Interestingly, the nanoflower co-localized with both Mycobacterium tuberculosis and Leishmania infantum within infected macrophages although these pathogens escape lysosomal degradation by affecting the phagocytotic pathway in very different manners. These results suggest an intriguing and overlooked potential application of DNA structures in targeted treatment of infectious diseases such as tuberculosis and leishmaniasis that are caused by pathogens that escape the human immune system by modifying macrophage biology.

Highly fluorescent morpholine naphthalimide deoxyuridine nucleotide for the detection of miRNA 24-3P through rolling circle amplification.

In this study we synthesized the nucleotide dUrkTP, a highly fluorescent naphthalimide deoxyuridine triphosphate that undergoes aggregation-induced emission (AIE). We incorporated and extended dUrkTP during the primer extension of DNA mediated by DNA polymerase, and also in the rolling circle amplification of DNA mediated by Phi29 polymerase. Accordingly, we could use this fluorescent nucleotide for the detection of microRNA 24-3P, a biomarker of porcine reproductive and respiratory syndrome virus. The direct labeling system obtained during rolling circle DNA amplification exhibited increased fluorescence, due to AIE of the dUrkTP residue upon gel formation, thereby allowing the detection of miRNA 24-3P. This direct labeling system facilitated the simple and inexpensive detection of miRNA 24-3P with high sensitivity (limit of detection: 3.58 fM) and selectivity.

Design of DNA rolling-circle templates with controlled fork topology to study mechanisms of DNA replication

Rolling-circle DNA amplification is a powerful tool employed in biotechnology to produce large from small amounts of DNA. This mode of DNA replication proceeds via a DNA topology that resembles a replication fork, thus also providing experimental access to the molecular mechanisms of DNA replication. However, conventional templates do not allow controlled access to multiple fork topologies, which is an important factor in mechanistic studies. Here we present the design and production of a rolling-circle substrate with a tunable length of both the gap and the overhang, and we show its application to the bacterial DNA-replication reaction.

Direct incorporation and extension of a fluorescent nucleotide through rolling circle DNA amplification for the detection of microRNA 24-3P

We designed and synthesized several fluorescent nucleotides from thiophene, anthracene and pyrene, which have different sizes, and screened their incorporation and extension capability during the rolling circle amplification of DNA. The thiophene-based fluorescent nucleotide (dUthioTP) could highly incorporate and extended into the rolling circle DNA product, while other fluorescent nucleotides (dUanthTP, and dUpyrTP) could not. This dUthioTP fluorescent nucleotide could be used for the detection of miRNA 24-3P, which is related PRRSV. This direct labeling system during rolling circle DNA amplification exhibited an increased fluorescence signal showing gel formation for the detection of miRNA 24-3P. This direct labeling system is a very simple and cost-efficient method for the detection miRNA 24-3P and also exhibited highly sensitive and selective detection properties.

Detection and characterization of tomato leaf curl New Delhi virus association with mosaic disease of ivy gourd (Coccinia grandis (L.) Voigt) in North India

Sixteen ivy gourd ( Coccinia grandis (L.) Voigt) plant samples showing severe mosaic symptoms were collected from New Delhi and Varanasi (Uttar Pradesh) in India. Begomovirus infection was confirmed by PCR using begomovirus-specific primers. Amplified PCR products (1.2 kb fragments) were cloned and the sequence was characterized. Based on sequence analysis, begomovirus associated with the majority of ivy gourd samples (16) was found to be a member of a bipartite begomovirus species, which is closely related to tomato leaf curl New Delhi virus (ToLCNDV). Therefore, two samples of ivy gourd, IVG1-ND and IVG2-Var, were selected for full-length genome (DNA-A and DNA-B-like sequence) amplification by the rolling circle DNA amplification (RCA) method. Sequence analysis performed using the Species Demarcation Tool (SDT) program revealed that they share 89.5-91.3% (IVG1-ND) and 93.4-96.8% (IVG2-Var) nucleotide (nt) identity with the DNA-A-like sequence of ToLCNDV isolated from cucurbits and chilli, respectively. The IVG1-ND and IVG2-Var isolates shared 90% nt identity among themselves, indicating that they are two different strains of ToLCNDV. Similarly, SDT analysis of the DNA-B-like sequence of IVG1-ND and IVG2-Var exhibited showed 82.7-93.3% nt identity with the DNA-B-like sequences of ToLCNDV infecting cucurbits. The recombination analysis of DNA-A and DNB-B-like sequences showed that the greater part of their genome most likely originated from previously reported begomoviruses that are known to infect chilli and cucurbits through recombination. https://doi.org/10.2298/ABS170616051V Received: June 16, 2017; Revised: November 16, 2017; Accepted: November 29, 2017; Published online: December 21, 2017 How to cite this article: Venkataravanappa V, Narasimha Reddy LRC, Saha S, Subbanna SK, Manem KR. Detection and characterization of tomato leaf curl New Delhi virus association with mosaic disease of ivy gourd ( Coccinia grandis (L.) Voigt) in North India. Arch Biol Sci. 2018;70(2):339-47.

Abstract 5571: Technologies for molecular epidemiology in cancer

Abstract Future success in molecular epidemiology depends on how quickly epidemiology adopts, optimizes and validates emerging technologies for molecular-scale analysis. In order to advance molecular epidemiology in cancer, the National Cancer Institute (NCI) supports the development of novel, next-generation, and cutting-edge molecular and cellular analysis technologies. Here we present past and present technology development efforts and their impact on cancer epidemiology. Some of the technologies, whose early-stage development was supported by the NCI, such as resequencing arrays, gene expression assay system, technologies which enable next generation sequencing, RNA preservative, rolling circle amplification of DNA, digital PCR system, functionalization of quantum dots, chromatin immunoprecipitation with next gen Sequencing, and multidimensional protein identification technology are now mainstay in basic and clinical research laboratories for genomic, epigenomic, transcriptomic and proteomic analysis. Additionally, these technologies have significantly contributed in identifying molecular determinants of cancer, and in assessing risk, disease progression and prognosis in large scale cancer epidemiology. Current awards support developing technologies for single cell analysis which can capture heterogeneity, simultaneous detection of multiple oncogenes, sensitive sequencing of geneomic/somatic mutations, targeted sequencing, rare variant detection, sequencing of ultralow-frequency mutations, epigenetic changes, kinome, multiplexed detection of microRNA, proteins, cytokines and metabolites, circulating tumor cells, and highthroughput protein-DNA interactions assessment. In addition, several novel biospecimen collection, preservation and processing technologies are also being supported with the potential to define the future of molecular epidemiology. Collectively, these technology development efforts supported by NCI provide a tremendous opportunity for molecular epidemiologists wishing to study archived samples collected in large-scale epidemiology efforts, and optimize and validate them for new molecular analyses. Citation Format: Rao L. Divi, Mukesh Verma, Anthony Dickherber. Technologies for molecular epidemiology in cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5571. doi:10.1158/1538-7445.AM2015-5571

Gateway-compatible vectors for high-throughput protein expression in pro- and eukaryotic cell-free systems

Although numerous techniques for protein expression and production are available the pace of genome sequencing outstrips our ability to analyze the encoded proteins. To address this bottleneck, we have established a system for parallelized cloning, DNA production and cell-free expression of large numbers of proteins. This system is based on a suite of pCellFree Gateway destination vectors that utilize a Species Independent Translation Initiation Sequence (SITS) that mediates recombinant protein expression in any in vitro translation system. These vectors introduce C or N terminal EGFP and mCherry fluorescent and affinity tags, enabling direct analysis and purification of the expressed proteins. To maximize throughput and minimize the cost of protein production we combined Gateway cloning with Rolling Circle DNA Amplification. We demonstrate that as little as 0.1ng of plasmid DNA is sufficient for template amplification and production of recombinant human protein in Leishmania tarentolae and Escherichia coli cell-free expression systems. Our experiments indicate that this approach can be applied to large gene libraries as it can be reliably performed in multi-well plates. The resulting protein expression pipeline provides a valuable new tool for applications of the post genomic era.

Complete genomic sequence of a new Human polyomavirus 9 strain with an altered noncoding control region.

A complete Human polyomavirus 9 (HPyV9) genome, designated HPyV9 UF-1, was amplified by rolling circle DNA amplification from DNA extracted from the peripheral blood mononuclear cells (PBMC) of an AIDS patient. The noncoding control (enhancer/promoter) region (NCCR) of HPyV9 UF-1 has one less AML-1a binding site and three more potential Sp1/GC box binding sites than the NCCRs of two previously described HPyV9 genomes. Nucleotide polymorphisms within the coding regions result in two amino acid differences in the deduced VP2 and VP3 proteins of HPyV9 UF-1 relative to those of the two previously described HPyV9 genomes. Exhaustive attempts to detect HPyV9 in DNA samples extracted from the PBMC of 40 healthy humans and 9 other AIDS patients were unsuccessful, highlighting the need for improved search strategies and optimal specimens for the detection of HPyV9 in humans.

Targeted and robust amplification of mitochondrial DNA in the presence of nuclear-encoded mitochondrial pseudogenes using Φ29 DNA polymerases.

Accidental co-amplification of nuclear-encoded mitochondrial pseudogenes (numts) in mitochondrial DNA (mtDNA) analyses is a common problem in biological and medical research alike [Yao et al., J Med Genet 45:769-772, 2008; Song et al., Proc Natl Acad Sci U S A 105:13486-13491, 2008]. A wealth of strategies have been developed to evade the contamination of mtDNA data sets with numts, but time- and cost-effective protocols of general applicability are scarce [Wolff et al., PLoS One 7:e37142, 2012]. The protocol presented here closes this gap by combining serial dilution with standard and rolling circle DNA amplification. This strategy leads to selective enrichment of mtDNA and removal of nuclear DNA (nuDNA) from template solutions, enabling mtDNA analyses in a virtually numt-free environment.

Detection of Nucleic Acid Targets Using Ramified Rolling Circle DNA Amplification: A Single Nucleotide Polymorphism Assay Model

BackgroundIsothermal amplification methods provide alternatives to PCR that may be preferable for some nucleic acid target detection tasks. Among current isothermal target detection methods, ramified rolling circle amplification (RAM) of single-stranded DNA circles that are formed by ligation of linear DNA probes (C-probes or padlock probes) offers a unique target detection system by linked primers and a simple amplification system that is unconstrained by the target’s sequence context. Earlier implementations of RAM-based target detection were reported to be limited by background noise, due in part to unligated C-probe in the amplification reaction. We show here that a target-detection system using a biotinylated target-capture probe together with automated bead-handling reduces or eliminates background amplification noise. We demonstrate the system’s performance by detection of a single-nucleotide polymorphism in human genomic DNA.MethodologyTarget detection by RAM entails hybridization and ligation of a C-probe, followed by amplification and RAM signal detection. We evaluated RAM target detection in genomic DNA using recognition of a human Factor V gene single nucleotide polymorphism (G1691A) as a model. Locus-specific C-probes were annealed and ligated to genomic DNAs that represent the 3 possible genotypes at this locus, then ligated C-probes were amplified by real time RAM. The majority of the steps in the assay were performed with a magnetic bead-based chemistry on an automated platform. We show that the specificity of C-probe ligation permits accurate genotyping of this polymorphism. The assay as described here eliminates some of the background noise previously described for C-probe ligation, RAM amplification assays.ConclusionThe methods and results presented here show that a combination of C-probe detection, automated sample processing, and isothermal RAM amplification provide a practical approach for detecting DNA targets in complex mixtures.

USE OF THE PROXIMITY LIGATION ASSAY TO DETECT IMMUNE COMPLEXES IN HISTOLOGIC SECTIONS

The Proximity Ligation Assay (PLA) combines elements of immunohistology and molecular biology to allow for in‐situ detection of specific proteins, protein modifications, and protein‐protein interactions. PLA is often used to study the interaction of signaling cascade phosphoproteins on cytospin preparations of cultured cells. We demonstrate that the PLA can be adapted to detect immune complexes in archived tissue sections. We established immune complexes in sections of H929 xenografts, which constitutively express human antibody kappa light chains, by incubating the sections with either mouse‐anti‐human light chain monoclonal or anti‐human IgG polyclonal antibodies. We then used multicolor immunofluorescence to confirm that both mouse and human antibodies specifically co‐localized to the H929 cells in the xenograft. Although multi‐color immunofluorescence indicates whether two proteins are located in the same general vicinity, it does not prove that the two proteins are bound to one another. In contrast, the PLA generates a fluorescent signal via rolling circle DNA amplification only when two proteins are bound to one another. Using the PLA, we confirmed that the mouse antibodies formed immune complexes with the constitutively expressed human antibodies in the cytoplasm of the H929 xenograft cells. PLA may be useful to detect the presence of immune complexes in both preclinical and clinical samples.

Fabrication of DNA polymer brush arrays by destructive micropatterning and rolling-circle amplification.

A method for fabricating DNA polymer brush arrays using photolithography and plasma etching followed by solid-phase enzymatic DNA amplification is reported. After attaching oligonucleotide primers to the surface of a glass coverslip, a thin layer of photoresist is spin-coated on the glass and patterned via photolithography to generate an array of posts in the resist. An oxygen-based plasma is then used to destroy the exposed oligonucleotide primers. The glass coverslip with the primer array is assembled into a microfluidic chip and DNA polymer brushes are synthesized on the oligonucleotide array by rolling-circle DNA amplification. We have demonstrated that the linear polymers can be rapidly synthesized in situ with a high degree of control over their density and length.

Identification of Diagnostic Biomarkers for Infection in Premature Neonates

Infection is a leading cause of neonatal morbidity and mortality worldwide. Premature neonates are particularly susceptible to infection because of physiologic immaturity, comorbidity, and extraneous medical interventions. Additionally premature infants are at higher risk of progression to sepsis or severe sepsis, adverse outcomes, and antimicrobial toxicity. Currently initial diagnosis is based upon clinical suspicion accompanied by nonspecific clinical signs and is confirmed upon positive microbiologic culture results several days after institution of empiric therapy. There exists a significant need for rapid, objective, in vitro tests for diagnosis of infection in neonates who are experiencing clinical instability. We used immunoassays multiplexed on microarrays to identify differentially expressed serum proteins in clinically infected and non-infected neonates. Immunoassay arrays were effective for measurement of more than 100 cytokines in small volumes of serum available from neonates. Our analyses revealed significant alterations in levels of eight serum proteins in infected neonates that are associated with inflammation, coagulation, and fibrinolysis. Specifically P- and E-selectins, interleukin 2 soluble receptor alpha, interleukin 18, neutrophil elastase, urokinase plasminogen activator and its cognate receptor, and C-reactive protein were observed at statistically significant increased levels. Multivariate classifiers based on combinations of serum analytes exhibited better diagnostic specificity and sensitivity than single analytes. Multiplexed immunoassays of serum cytokines may have clinical utility as an adjunct for rapid diagnosis of infection and differentiation of etiologic agent in neonates with clinical decompensation.

Artificial site-specific DNA-nicking system based on common restriction enzyme assisted by PNA openers.

We report on the peptide nucleic acid (PNA)-directed design of a DNA-nicking system that enables selective and quantitative cleavage of one strand of duplex DNA at a designated site, thus mimicking natural nickases and significantly extending their potential. This system exploits the ability of pyrimidine PNAs to serve as openers for specific DNA sites by invading the DNA duplex and exposing one DNA strand for oligonucleotide hybridization. The resultant secondary duplex can act as a substrate for a restriction enzyme, which ultimately creates a nick in the parent DNA. We demonstrate that several restriction enzymes of different types could be successfully used in the PNA-assisted system we developed. Importantly, the enzyme cleavage efficiency is basically not impaired on such artificially generated substrates, compared with the efficiency on regular DNA duplexes. Our design originates a vast class of semisynthetic rare-cleaving DNA nickases, which are essentially absent at present. In addition, we show that the site-specific PNA-assisted nicking of duplex DNA can be engaged in a rolling-circle DNA amplification (RCA) reaction. This new RCA format demonstrates the practical potential of the novel biomolecular tool we propose for DNA technology and DNA diagnostics.

Visualization of oligonucleotide probes and point mutations in interphase nuclei and DNA fibers using rolling circle DNA amplification.

Rolling circle amplification (RCA) is a surface-anchored DNA replication reaction that can be exploited to visualize single molecular recognition events. Here we report the use of RCA to visualize target DNA sequences as small as 50 nts in peripheral blood lymphocytes or in stretched DNA fibers. Three unique target sequences within the cystic fibrosis transmembrane conductance regulator gene could be detected simultaneously in interphase nuclei, and could be ordered in a linear map in stretched DNA. Allele-discriminating oligonucleotide probes in conjunction with RCA also were used to discriminate wild-type and mutant alleles in the cystic fibrosis transmembrane conductance regulator, p53, BRCA-1, and Gorlin syndrome genes in the nuclei of cultured cells or in DNA fibers. These observations demonstrate that signal amplification by RCA can be coupled to nucleic acid hybridization and multicolor fluorescence imaging to detect single nucleotide changes in DNA within a cytological context or in single DNA molecules. This provides a means for direct physical haplotyping and the analysis of somatic mutations on a cell-by-cell basis.

Rolling circle amplification of DNA immobilized on solid surfaces and its application to multiplex mutation detection

A new method of amplifying short DNA molecules immobilized on a solid support has been developed. This method uses a solid-phase rolling circle replication reaction, termed rolling circle amplification (RCA). The probe consists of a single-stranded DNA primer anchored at the 5′ terminus to a solid support and a single stranded DNA template hybridized to the immobilized primer. Here, DNA ligase was used to circularize the template, and DNA polymerase I was used to extend the immobilized primer in a rolling circle replication reaction. This method was used to identify a known polymorphism in BRCA1 exon 5. These results demonstrate that RCA offers considerable promise to facilitate effective mutation screening of DNA using a solid-phase format.