The single-nucleotide primer extension (SNuPE) method for the multiplex detection of various DNA sequences: from detection of point mutations to microbial ecology

Abstract

Methods based on SNuPE (single-nucleotide primer extension) have become invaluable tools for the rapid and highly specific detection of point mutations and single-nucleotide polymorphisms in the field of human genetics. In the primer extension reaction, a DNA polymerase is used to label a specific primer hybridized to the target sequence by incorporating a single labelled ddNTP (dideoxynucleotide). This labelling provides not only information about the complementary nucleotide of interest in the opposite strand but also a semiquantitative analysis of the sequence targeted by the primer. Since several subdisciplines of microbiology increasingly require cultivation-independent molecular screening tools for elucidating differences between either strains or community structures based on sequence variations of marker genes, SNuPE offers a promising alternative to the existing tool box. The present review describes the method in detail and reports the state-of-the-art applications of this technique both in the field of nucleic acid detections in human genetics and in microbiology.

Enzymological description of multitemplate PCR—Shrinking amplification bias by optimizing the polymerase–template ratio

“…Single nucleotide primer extension (SNuPE) is a variant in which a primer anneals to the target sequence with its 3 0 end immediately adjacent to the polymorphic site. The primer is extended by DNA polymerase using fluorescent dideoxynucleotides, and the identity of the polymorphic site is revealed by identification of the incorporated fluorescent dideoxynucleotide (Nikolausz et al, 2009).…”

Temporal Development of the Infant Gut Microbiota in Immunoglobulin E-Sensitized and Nonsensitized Children Determined by the GA-Map Infant Array

“…The main difference between the GA-map infant array and alternative 16S rRNA gene array approaches (19,23) is the use of highly specific single nucleotide primer extension (SNuPE) probes for target/nontarget discrimination (17,27). The high specificity of the SNuPE assay is obtained by the combined fidelity provided by DNA polymerase-based incorporation of a fluorescently labeled dideoxynucleotide and target hybridization (16,31).…”

Personalized treatment in the eradication therapy for Helicobacter pylori

“…As in previous reports, EMs resulted in a low eradication success rate caused by the lack of an increase in blood PPI levels, which is supported by this study. Therefore, modification of PPI dosing or drugs should be considered based on CYP2C19 genotyping before treatment (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15).…”

“…The CAM resistance gene and the CYP2C19 gene polymorphisms were analyzed by the SNuPE method (15,21). First, the region including the polymorphisms was amplified with 1 μg of DNA extracted from gastric mucosa and a total of 10 μl constituting of 5 μl of Multiplex PCR Mix (Qiagen) and each primer at 0.2 μM.…”

“…CYP2C19 which is related to the metabolism of PPI also has gene polymorphisms that have been reported to be related to enzyme activity. More than 20 alleles are present in CYP2C19 polymorphisms, but because only 3 types have been reported in the Japanese population, we developed a method of analysis for these 3 alleles using the SNuPE method (15)(16)(17)(18)(19)(20).…”