Template For Rolling Circle Amplification Research Articles

First Detection of Cotton leaf curl Burewala virus and Cognate Cotton leaf curl Multan betasatellite and Gossypium darwinii symptomless alphasatellite in Symptomatic Luffa cylindrica in Pakistan.

Cotton leaf curl disease (CLCuD) is the major plant viral constraint to cotton production on the Indian subcontinent (2). CLCuD is primarily caused by begomovirus, Cotton leaf curl Burewala virus (CLCuBuV), and Cotton leaf curl Multan betasatellite (CLCuMB). During 2011 in Burewala, Pakistan, plants in a production field of Luffa cylindrica (Ghia tori) were infested with the whitefly Bemisia tabaci (Genn.), and ~60% of the plants exhibited leaf curling and stunting symptoms, reminiscent of those caused by begomoviruses (Geminiviridae). Total DNA was extracted from five different symptomatic leaf samples using the CTAB method (1), and extracts were analyzed by Southern blot hybridization. As a probe, we used a 1.1-kbp fragment of CLCuBuV and a positive signal was obtained from all five samples. Total DNA was used as template for rolling circle amplification (RCA) using the TempliPhi DNA Amplification Kit (GE Healthcare, Little Chalfont, United Kingdom). The amplified RCA products were digested with EcoRI, and the resulting ~2.7-kbp fragments from each isolate were directionally cloned into the EcoRI digested, pGEM-3Zf+ (Promega, Madison, WI) plasmid vector. PCR was used to amplify the prospective, associated betasatellite and alphasatellite molecules using the primers BetaF5'-GGTACCGCCGGAGCTTAGCWCKCC-3' and BetaR5'-GGTACCGTAGCTAAGGCTGCTGCG-3', and AlphaF5'-AAGCTTAGAGGAAACTAGGGTTTC-3' and AlphaR5'-AAGCTTTTCATACARTARTCNCRDG-3', respectively. The putative satellite amplicons, at ~1.4 kbp each were cloned in the plasmid vector pGEMT-Easy (Promega, Madison, WI) and sequenced. BLASTn comparisons of the apparently full-length begomoviral genomes, at 2,753 nt, against the NCBI database revealed that all five isolates were most closely related to CLCuBuV (FR750321). In addition, one each of beta- and alpha-satellite were amplified from all five samples at 1,393 and 1,378 bases, respectively. The beta- and alpha-satellites were most closely related to CLCuMB (HE985228) and the Gossypium darwinii symptomless alphasatellite (GDaSA) (FR877533), respectively. Pairwise sequence comparisons of the top 10 BLASTn hits using MEGA5 indicated that the helper begomovirus shared 99.9% identity with CLCuBuV (FR750321), the most prevalent helper virus currently associated with the leaf curl complex in Pakistan. Based on the ICTV demarcation for begomoviral species at <89%, it is considered a variant of CLCuBuV. The resultant beta- and alpha-satellite sequences were 98.1% and 97.8% identical to CLCuMB (HE985228) and GDaSA (FR877533), respectively, and are the most prevalent satellites associated with the CLCuD complex in Pakistan and India (2). To our knowledge, this is first report of the CLCuBuV-CLCuMB-GDaSA complex infecting a cucurbitaceous species, and the first report of L. cylindrica as a host of the CLCuD complex. This discovery of CLCuBuV and associated satellites in a cucurbitaceous host that is widely grown in Pakistan and India where this complex infects cotton indicates that the host range of CLCuBuV is broader than expected. This new information will aid in better understanding of cotton leaf curl disease epidemiology in the current epidemic areas.

Production of Single-Stranded DNAs by Self-Cleavage of Rolling-Circle Amplification Products

DNA molecules that encode a small, high-speed self-hydrolyzing deoxyribozyme are used as templates for rolling circle amplification (RCA) to produce single-stranded DNAs (ssDNAs) of single- and multiple-unit lengths. Including self-cleaving deoxyribozymes in RCA products can generate large amounts of ssDNAs with defined sequence and length as well as precise termini. We also demonstrate the use of this method to efficiently generate ssDNA size markers by using deoxyribozyme reaction conditions that permit partial processing.

Single-Molecule DNA Patterning and Detection by Padlock Probing and Rolling Circle Amplification in Microchannels for Analysis of Small Sample Volumes

The rolling circle amplification (RCA) is a versatile DNA amplification method in which a DNA molecule is amplified using a single DNA primer, allowing the product to be counted as a single dot. Circular templates for RCA can arise from padlock probes in highly specific DNA target-mediated ligation reactions. However, improvement of detection efficiency represents an important challenge. In homogeneous assays, the detection efficiency is generally only under 0.1%, mainly because the sample volume is too large compared with the detection volume. Here, we used microchannel surfaces in a glass microchip for DNA detection in small volume samples. First, DNA patterning on glass surfaces in microchannels was demonstrated using chemical surface patterning by UV light. By using a photochemical reaction, we realized DNA patterning in a closed space. Second, RCA was demonstrated using dilutions of target molecules, and a calibration curve was obtained. The highest detection efficiency was 22.5% by virtue of the reduced sample volumes from several hundred microliters to 5.0 nL. Accordingly, a countable number of DNA molecules was successfully detected. This method is suitable for analysis of very small volume samples such as single cells, especially by using extended-nanochannels with dimensions of 10-1000 nm.

Diffractometric detection of proteins using microbead-based rolling circle amplification.

We present a robust, sensitive, fluorescent- or radiolabel-free self-assembled optical diffraction biosensor that utilizes rolling circle amplification (RCA) and magnetic microbeads as a signal enhancement method. An aptamer-based sandwich assay was performed on microcontact-printed streptavidin arranged in 15 microm wide alternating lines and could specifically capture and detect platelet-derived growth factor B-chain (PDGF-BB). An aptamer served as a template for the ligation of a padlock probe, and the circularized probe could in turn be used as a template for RCA. The concatameric RCA product hybridized to biotinylated oligonuclotides which then captured streptavidin-labeled magnetic beads. In consequence, the signal from the captured PDGF-BB was amplified via the concatameric RCA product, and the diffraction gratings on the printed areas produced varying intensities of diffraction modes. The detected diffraction intensity and the density of the microbeads on the surface varied as a function of PDGF-BB concentration. Our results demonstrate a robust biosensing platform that is easy to construct and use and devoid of fluorescence microscopy. The self-assembled bead patterns allow both a visual analysis of the molecular binding events under an ordinary bright-field microscope and serve as a diffraction grating biosensor.

Fluorescence-based detection of short DNA sequences under non-denaturing conditions

The ability of peptide nucleic acid (PNA) to open up duplex DNA in a highly sequence-specific manner makes it possible to detect short DNA sequences on the background of or within genomic DNA under non-denaturing conditions. To do so, chosen marker sites in double-stranded DNA are locally opened by a pair of PNA openers, thus transforming one strand within the target region (20–30 bp) into the single-stranded form. Onto this accessible DNA sequence a circular oligonucleotide probe is assembled, which serves as a template for rolling circle amplification (RCA). Both homogeneous and heterogeneous assay formats are investigated, as are different formats for fluorescence-based amplicon detection. Our recent data with immobilized analytes suggest that marker sequences in plasmid and bacterial chromosomal DNA can be successfully detected.

Using a Deoxyribozyme Ligase and Rolling Circle Amplification To Detect a Non-nucleic Acid Analyte, ATP

An allosteric ribozyme (aptazyme) has been used to transduce the binding of a small organic analyte (ATP) into the ligation of a circular template for rolling circle amplification (RCA). An ATP-activated deoxyribozyme ligase was immobilized on a glass slide and, upon addition of ATP, catalyzed the ligation of a circular padlock probe. The ligated products could be directly amplified and visualized via RCA. The coupled reaction exhibited could detect as little as 1 muM of ATP and could discriminate against structurally similar nucleotides such as GTP, CTP, and UTP. Cooperative ATP activation of the deoxyribozyme was faithfully mimicked by RCA, yielding an amplified "switch" that was responsive to ATP concentration.