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Selectivity of Enzymatic Conversion of Oligonucleotide Probes during Nucleotide Polymorphism Analysis of DNA.

ABSTRACT: The analysis of DNA nucleotide polymorphisms is one of the main goals of DNA diagnostics. DNA-dependent enzymes (DNA polymerases and DNA ligases) are widely used to enhance the sensitivity and reliability of systems intended for the detection of point mutations in genetic material. In this article, we have summarized the data on the selectiveness of DNA-dependent enzymes and on the structural factors in enzymes and DNA which influence the effectiveness of mismatch discrimination during enzymatic conversion of oligonucleotide probes on a DNA template. The data presented characterize the sensitivity of a series of DNA-dependent enzymes that are widely used in the detection of noncomplementary base pairs in nucleic acid substrate complexes. We have analyzed the spatial properties of the enzyme-substrate complexes. These properties are vital for the enzymatic reaction and the recognition of perfect DNA-substrates. We also discuss relevant approaches to increasing the selectivity of enzyme-dependent reactions. These approaches involve the use of modified oligonucleotide probes which "disturb" the native structure of the DNA-substrate complexes.

SUBMITTER: Vinogradova OA

PROVIDER: S-EPMC3347538 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Selectivity of Enzymatic Conversion of Oligonucleotide Probes during Nucleotide Polymorphism Analysis of DNA.

Vinogradova O A OA Pyshnyi D V DV

Acta naturae 20100401 1

The analysis of DNA nucleotide polymorphisms is one of the main goals of DNA diagnostics. DNA-dependent enzymes (DNA polymerases and DNA ligases) are widely used to enhance the sensitivity and reliability of systems intended for the detection of point mutations in genetic material. In this article, we have summarized the data on the selectiveness of DNA-dependent enzymes and on the structural factors in enzymes and DNA which influence the effectiveness of mismatch discrimination during enzymatic ...[more]

PMID: 22649627

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Project description:Summary In this work, we developed a simple and robust assay to rapidly detect SNPs in nucleic acid samples. Our approach combines loop-mediated isothermal amplification (LAMP)-based target amplification with fluorescent probes to detect SNPs with high specificity. A competitive “sink” strand preferentially binds to non-SNP amplicons and shifts the free energy landscape to favor specific activation by SNP products. We demonstrated the broad utility and reliability of our SNP-LAMP method by detecting three distinct SNPs across the human genome. We also designed an assay to rapidly detect highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants from crude biological samples. This work demonstrates that competitive SNP-LAMP is a powerful and universal method that could be applied in point-of-care settings to detect any target SNP with high specificity and sensitivity. We additionally developed a publicly available web application for researchers to design SNP-LAMP probes for any target sequence of interest. Graphical abstract Highlights • A simple and rapid assay to detect specific SNPs in complex nucleic acid samples• Approach leverages competitive probes to specifically detect SNP over non-SNP sequences• Demonstrated utility across diverse targets and sample types• Web application for researchers to design SNP-LAMP probes for any target of interest Motivation Single-nucleotide polymorphisms (SNPs) are the most common source of genetic variation between individuals and have implications in human disease, pathogen drug resistance, and agriculture. SNPs are often detected using DNA sequencing or PCR, both of which can be difficult to deploy for on-site testing or in low-resource settings. There is a need for new SNP detection methods that can be performed rapidly without advanced laboratory equipment or a cold supply chain. Detecting single-nucleotide polymorphisms (SNPs) is important for understanding human disease, the emergence of pathogen variants, and the application of genetic agricultural breeding programs. Hyman et al. develop a simple and rapid assay to detect SNPs in complex nucleic acid samples. A web-based tool allows researchers to design custom SNP probes.

Dataset Information

Selectivity of Enzymatic Conversion of Oligonucleotide Probes during Nucleotide Polymorphism Analysis of DNA.

Publications

Selectivity of Enzymatic Conversion of Oligonucleotide Probes during Nucleotide Polymorphism Analysis of DNA.

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