Large Number Of Oligonucleotides Research Articles

OligoPrime: An Information System for Oligonucleotide Management.

With the increasing number of molecular biology techniques, large numbers of oligonucleotides are frequently involved in individual research projects. Thus, a dedicated electronic oligonucleotide management system is expected to provide several benefits such as increased oligonucleotide traceability, facilitated sharing of oligonucleotides between laboratories, and simplified (bulk) ordering of oligonucleotides. Herein, we describe OligoPrime, an information system for oligonucleotide management, which presents a computational support for all steps in an oligonucleotide lifecycle, namely, from its ordering and storage to its application, and disposal. OligoPrime is easy to use since it is accessible via a web browser and does not require any installation from the end user’s perspective. It allows filtering and search of oligonucleotides by various parameters, which include the exact location of an oligonucleotide, its sequence, and availability. The oligonucleotide database behind the system is shared among the researchers working in the same laboratory or research group. Users might have different roles which define the access permissions and range from students to researchers and primary investigators. Furthermore, OligoPrime is easy to manage and install and is based on open-source software solutions. Its code is freely available at https://github.com/OligoPrime. Moreover, an implementation of OligoPrime, which can be used for testing is available at http://oligoprime.xyz/. To our knowledge, OligoPrime is the only software solution dedicated specifically to oligonucleotide management. We strongly believe that it has a large potential to enhance the transparency of use and to simplify the management of oligonucleotides in academic laboratories and research groups.

An ultrasensitive fluorescence assay for protein detection by hybridization chain reaction-based DNA nanotags

An ultrasensitive fluorescence method for determination of protein is developed based on hybridization chain reaction (HCR). In this assay, the streptavidin-magnetic nanobeads were conjugated to biotinylated initiators and biotinylated anti-IgG. In the presence of human IgG, the magnetic nanobeads were fixed on the substrate and the carried initiators propagated the chain reaction of hybridization to form the nicked polymers. Because the nanobead probe carries with a large number of oligonucleotides per protein binding event, there is obvious amplification in the nicked polymers. Then, numerous SYBR Green I molecules were intercalated into the grooves of the long dsDNA polymers, generating a substantially apparent increase in the corresponding fluorescence intensity. With HCR amplification and magenetic nanobead to preamplify the fluorescence signal and reduce the background signal, the detection limit of this assay was 14aM. Compared with the reported protein detection methods, our method exhibited ultrahigh sensitivity. In addition, the proposed method possessed excellent selectivity and low matrix effect. What is more, the assay was also studied for clinical application in human serum with a satisfactory and reliable result.

MNAzyme qPCR with Superior Multiplexing Capacity

MNAzymes (nucleic acid enzymes formed from multiple partial enzymes) can be linked to PCR to provide a highly specific method for target detection and quantification. We investigated the feasibility of multiplexing MNAzyme quantitative PCR (qPCR) methods. We combined MNAzyme components with PCR primers and standard qPCR reagents to perform MNAzyme qPCR and reverse-transcription qPCR (RT-qPCR) assays with a set of universal reporter probes. Assays were performed on single targets and in multiplex formats that combined up to 5 different targets in a single reaction. A comparison of 3 targets amplified in single and triplex formats showed no significant differences with respect to detection limit or amplification efficiency. Likewise, we successfully converted single-target assays for 11 transcripts of interest to triplex assays containing 2 reference transcripts without having to optimize or modify the conditions. A quintuplex RT-qPCR that simultaneously quantified 5 transcripts with 5 universal probes produced high amplification efficiencies and r(2) values for all transcripts. Despite the large numbers of oligonucleotides in the reactions, we observed no false-positive signals, owing to the requirement of 4 target-specific binding events to produce a signal. A quadruplex assay that combined MNAzymes with methylation-specific PCR to measure epigenetic biomarkers of prostate cancer was capable of detecting a single methylated DNA allele in a background of 1000-10 000 unmethylated alleles. The MNAzyme qPCR was compatible with a rapid-cycling protocol. MNAzymes offer a flexible and unique approach to qPCR that is specific, sensitive, and easily multiplexed. The universal nature of MNAzyme reporter probes removes the need for target-specific probes, thereby making the development of new assays easier and cheaper.

Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins.

An ultrasensitive method for detecting protein analytes has been developed. The system relies on magnetic microparticle probes with antibodies that specifically bind a target of interest [prostate-specific antigen (PSA) in this case] and nanoparticle probes that are encoded with DNA that is unique to the protein target of interest and antibodies that can sandwich the target captured by the microparticle probes. Magnetic separation of the complexed probes and target followed by dehybridization of the oligonucleotides on the nanoparticle probe surface allows the determination of the presence of the target protein by identifying the oligonucleotide sequence released from the nanoparticle probe. Because the nanoparticle probe carries with it a large number of oligonucleotides per protein binding event, there is substantial amplification and PSA can be detected at 30 attomolar concentration. Alternatively, a polymerase chain reaction on the oligonucleotide bar codes can boost the sensitivity to 3 attomolar. Comparable clinically accepted conventional assays for detecting the same target have sensitivity limits of approximately 3 picomdar, six orders of magnitude less sensitive than what is observed with this method.

Optimal sequencing by hybridization in rounds.

Sequencing by hybridization (SBH) is a method for sequencing DNA. The Watson-Crick complementarity of DNA can be used to determine whether the DNA contains an oligonucleotide substring. A large number of oligonucleotides can be arranged on an array (SBH chip). A combinatorial method is used to construct the sequence from the collection of probes that occur in it. We develop an idea of Margaritis and Skiena and propose an algorithm that uses a series of small SBH chips to sequence long strings. The total number of probes used by our method matches the information theoretical lower bound up to a constant factor.

In situ hybridization with oligonucleotide probes

Publisher Summary In situ hybridization (ISH) is an important method for tracing the regional and cellular sites of gene expression (mRNA distribution) within a tissue. This method comes into its own when applied to the intermingled cell types of the brain. ISH allows the particular cell type in which the mRNA is contained to be marked out from others. The technique is simple—a nucleic-acid probe, tagged with either radiolabeled nucleotides or molecules allowing calorimetric/light detection, is applied to a tissue section. The probe searches out and finds (hybridizes to) its corresponding mRNA in the cells and forms a probe-mRNA double helix. After the excess probe has been washed away, the section is either exposed for autoradiography or processed histochemically to reveal the sites on the section targeted by the probe. ISH with oligonucleotides needs less hands-on time than other ISH methods. Large numbers of oligonucleotides (e.g., up to 20 at one time) and sections (e.g., 100) can be handled at once. The method is well suited to large-scale studies that, for example, compare the expression of all members of a gene family or map the expression of novel gene sets.