Single Nucleotide Discrimination Research Articles

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MicroRNAs (miRNAs) are relatively short, 22nt～, and endogenous non-coding RNAs. It is known to act as a sequence specific regulator of post-transcriptional gene expression in many eukaryotes, most of the time with some sequence mismatches against target genes. Recently, a couple of miRNAs have been shown to be involved in oncogenic pathway, development, or cell differentiation. miRNAs are now getting attention to its diverse regulatory and catalytic functions.Although many of the miRNA sequences have been registered in the public databases and their target genes are predicted by computational analysis, the empirical verification has to be done. One of the difficulties to study the miRNA function will be that its structural and regulatory characteristics.In order to overcome this difficulty and utilize miRNA as an experimental tool, we investigated a system to detect, knockdown, and / or overexpress miRNAs. First, we adopted bicyclic nucleic acid, Locked Nucleic Acid (LNA), to improve affinity and single nucleotide discrimination. Next, we established the system to introduce synthetic oligos or RNAs expressed from lentiviral vectors to cells. A novel approach based on these principles on miRNA and its pathway analysis will be discussed.

Dynamic assembly of primers on nucleic acid templates

A strategy is presented that uses dynamic equlibria to assemble in situ composite DNA polymerase primers, having lengths of 14 or 16 nt, from DNA fragments that are 6 or 8 nt in length. In this implementation, the fragments are transiently joined under conditions of dynamic equilibrium by an imine linker, which has a dissociation constant of ∼1 μM. If a polymerase is able to extend the composite, but not the fragments, it is possible to prime the synthesis of a target DNA molecule under conditions where two useful specificities are combined: (i) single nucleotide discrimination that is characteristic of short oligonucleotide duplexes (four to six nucleobase pairs in length), which effectively excludes single mismatches, and (ii) an overall specificity of priming that is characteristic of long (14 to 16mers) oligonucleotides, potentially unique within a genome. We report here the screening of a series of polymerases that combine an ability not to accept short primer fragments with an ability to accept the long composite primer held together by an unnatural imine linkage. Several polymerases were found that achieve this combination, permitting the implementation of the dynamic combinatorial chemical strategy.

Designing siRNA That Distinguish between Genes That Differ by a Single Nucleotide

Small interfering RNAs (siRNAs), the guides that direct RNA interference (RNAi), provide a powerful tool to reduce the expression of a single gene in human cells. Ideally, dominant, gain-of-function human diseases could be treated using siRNAs that specifically silence the mutant disease allele, while leaving expression of the wild-type allele unperturbed. Previous reports suggest that siRNAs can be designed with single nucleotide specificity, but no rational basis for the design of siRNAs with single nucleotide discrimination has been proposed. We systematically identified siRNAs that discriminate between the wild-type and mutant alleles of two disease genes: the human Cu, Zn superoxide dismutase (SOD1) gene, which contributes to the progression of hereditary amyotrophic lateral sclerosis through the gain of a toxic property, and the huntingtin (HTT) gene, which causes Huntington disease when its CAG-repeat region expands beyond approximately 35 repeats. Using cell-free RNAi reactions in Drosophila embryo lysate and reporter assays and microarray analysis of off-target effects in cultured human cells, we identified positions within an siRNA that are most sensitive to mismatches. We also show that purine:purine mismatches imbue an siRNA with greater discriminatory power than other types of base mismatches. siRNAs in which either a G:U wobble or a mismatch is located in the “seed” sequence, the specialized siRNA guide region responsible for target binding, displayed lower levels of selectivity than those in which the mismatch was located 3′ to the seed; this region of an siRNA is critical for target cleavage but not siRNA binding. Our data suggest that siRNAs can be designed to discriminate between the wild-type and mutant alleles of many genes that differ by just a single nucleotide.

Tuning Single Nucleotide Discrimination in Polymerase Chain Reactions (PCRs): Synthesis of Primer Probes Bearing Polar 4′‐C‐Modifications and Their Application in Allele‐Specific PCR

There are many methods available for the detection of nucleotide variations in genetic material. Most of these methods are applied after amplification of the target genome sequence by the polymerase chain reaction (PCR). Many efforts are currently underway to develop techniques that can detect single nucleotide variations in genes either by means of, or without the need for, PCR. Allele-specific PCR (asPCR), which reports nucleotide variations based on either the presence or absence of a PCR-amplified DNA product, has the potential to combine target amplification and analysis in one single step. The principle of asPCR is based on the formation of matched or mismatched primer-target complexes by using allele-specific primer probes. PCR amplification by a DNA polymerase from matched 3'-primer termini proceeds, whereas a mismatch should obviate amplification. Given the recent advancements in real-time PCR, this technique should, in principle, allow single nucleotide variations to be detected online. However, this method is hampered by low selectivity, which necessitates tedious and costly manipulations. Recently, we reported that the selectivity of asPCR can be significantly increased through the employment of chemically modified primer probes. Here we report further significant advances in this area. We describe the synthesis of various primer probes that bear polar 4'-C-modified nucleotide residues at their 3' termini, and their evaluation in real-time asPCR. We found that primer probes bearing a 4'-C-methoxymethylene modification have superior properties in the discrimination of single nucleotide variations by PCR.

Superb Nucleotide Discrimination by a Novel On/Off Switch for DNA Polymerization and Its Applications

With the use of polymerases having 3' to 5' exonuclease activity and 3' phosphorothioate-modified allele-specific primers, we recently devised a SNP-operated on/off switch controlling DNA polymerization. One advantage of this novel on/off switch is its adaptability to arrayed primer extension. To further expand its application in genetic analysis, this new on/off switch was evaluated in discrimination of the match/mismatch status of single nucleotides upstream from the primer 3' terminal. A set of seven amplicons was developed with the templates differing from each other by a single nucleotide. Using this set of amplicons, the new on/off switch was shown to be able to efficiently discriminate single nucleotide polymorphisms from the primer 3' terminus to the -6 position from the primer 3' terminus. These data, illustrating the broad single nucleotide discrimination ability of this novel on/off switch, explain why the SNP-operated on/off switch is powerful in SNP analysis, and also indicate useful applications to genetic analysis additional to SNP assay. First, these data broaden the application of the novel on/off switch in the analysis of mutations other than SNPs. Second, it raises a nucleotide-walking algorithm suitable for de novo array-based sequencing analysis.

Colloidal Nanoparticles from Poly(N-isopropylacrylamide)-graft-DNA for Single Nucleotide Discrimination Based on Salt-induced Aggregation: Extension to Long Target DNA

Abstract The DNA-linked colloidal nanoparticle was prepared through the phase transition of the amphiphilic copolymer consisting of DNA as the hydrophilic part and thermo-responsive poly(N-isopropylacrylamide) as the hydrophobic part. We have succeeded in detecting the single nucleotide difference of 39-mer target DNA by using a salt-induced aggregation of the particles.

Different Applications of Polymerases With and Without Proofreading Activity in Single-Nucleotide Polymorphism Analysis

With the completion of the human genome project, single-nucleotide polymorphisms (SNPs) have become the focus of intense study in biomedical research. Polymerase-mediated primer extension has been employed in a variety of SNP assays. However, these SNP assays using polymerase without proofreading function are compromised by their low reliability. Using a newly developed short amplicon harboring restriction enzyme site, EcoR-I, we were able to compare the single-base discrimination abilities of polymerases with and without proofreading function in primer extension in a broad range of annealing temperatures. Thermodynamic analysis demonstrated a striking single-nucleotide discrimination ability of polymerases with proofreading function. Using unmodified 3′-end allele-specific primers, only template-dependent products were generated by polymerase with proofreading activity. This powerful single-base discrimination ability of exo+ polymerases was further evaluated in primer extension using three types of 3′ terminally modified allele-specific primers. As compared with the poor fidelity in primer extension of polymerases lacking 3′ exonuclease activity, this study provides convincing evidence that the use of proofreading polymerases in combination with 3′-end modified allele-specific primers can be a powerful new strategy for the development of SNP assays.

Biochemical basis of DNA replication fidelity.

DNA polymerase is the critical enzyme maintaining genetic integrity during DNA replication. Individual steps in the replication process that contribute to DNA synthesis fidelity include nucleotide insertion, exonucleolytic proofreading, and binding to and elongation of matched and mismatched primer termini. Each process has been investigated using polyacrylamide gel electrophoresis (PAGE) to resolve 32P-labeled primer molecules extended by polymerase. We describe how integrated gel band intensities can be used to obtain site-specific velocities for addition of correct and incorrect nucleotides, extending mismatched compared to correctly matched primer termini and measuring polymerase dissociation rates and equilibrium DNA binding constants. The analysis is based on steady-state "single completed hit conditions", where polymerases encounter many DNA molecules but where each DNA encounters an enzyme at most once. Specific topics addressed include nucleotide misinsertion, mismatch extension, exonucleolytic proofreading, single nucleotide discrimination using PCR, promiscuous mismatch extension by HIV-1 and AMV reverse transcriptases, sequence context effects on fidelity and polymerase dissociation, structural and kinetic properties of mispairs relating to fidelity, error avoidance mechanisms, kinetics of copying template lesions, the "A-rule" for insertion at abasic template lesions, an interesting exception to the "A-rule", thermodynamic and kinetic determinants of base pair discrimination by polymerases.

Notes of shipbuilding and the construction of machinery in New York and vicinity

This study reports the development of an on-chip enzyme-mediated primer extension process based on a microfluidic device with microbeads array for single-nucleotide discrimination using quantum dots as labels. The functionalized microbeads were independently introduced into the arrayed chambers using the loading chip slab. A single channel was used to generate weir structures to confine the microbeads and make the beads array accessible by microfluidics. The applied allele-specific primer extension method employed a nucleotide-degrading enzyme (apyrase) to achieve specific single-nucleotide detection. Based on the apyrase-mediated allele-specific primer extension with quantum dots as labels, on-chip single-nucleotide discrimination was demonstrated with high discrimination specificity and sensitivity (0.5 pM, signal/noise > 3) using synthesized target DNA. The chip-based signal enhancement for single-nucleotide discrimination resulted in 200 times higher sensitivity than that of an off-chip test. This microfluidic device successfully achieved simultaneous detection of two disease-associated single-nucleotide polymorphism sites using polymerase chain reaction products as target. This apyrase-mediated microfluidic primer extension approach combines the rapid binding kinetics of homogeneous assays of suspended microbeads array, the liquid handling capability of microfluidics, and the fluorescence detection sensitivity of quantum dots to provide a platform for single-base analysis with small reagent consumption, short assay time, and parallel detection.