Genotyping of Single-Nucleotide Polymorphisms by Primer Extension Reaction in a Dry-Reagent Dipstick Format

Abstract

The primer extension (PEXT) reaction is the most widely used approach to genotyping of single-nucleotide polymorphisms (SNPs). Current methods for analysis of PEXT reaction products are based on electrophoresis, fluorescence resonance energy transfer, fluorescence polarization, pyrosequencing, mass spectrometry, microarrays, and spectrally encoded microspheres. We report the first dry-reagent dipstick method that enables rapid visual detection of PEXT products without instrumentation. The method is applied to the analysis of six SNPs in the mannose-binding lectin gene (MBL2). The genomic region that spans each SNP of interest is amplified by PCR. Two primer extension reactions are performed with allele-specific primers (for one or the other variant nucleotide), which contain an oligo(dA) segment at the 5'-end. Biotin-dUTP is incorporated in the extended strand. The product is applied to the strip followed by immersion in the appropriate buffer. As the DNA moves along the strip by capillary action, it hybridizes with oligo(dT)-functionalized gold nanoparticles, such that only extended products are captured by immobilized streptavidin at the test zone, generating a red line. A second red line is formed at the control zone of the strip by hybridization of the nanoparticles with immobilized oligo(dA). The dipstick test is complete within 10 min. We analyzed six SNPs of the mannose-binding lectin gene (MBL2) using genomic DNA from 27 patients, representing a total of 74 variant nucleotide positions. Patient genotypes showed 100% concordance with direct DNA sequencing data. The described PEXT-dipstick assay is rapid and highly accurate; it does not require specialized instrumentation or highly trained technical personnel. It is appropriate for a diagnostic laboratory where a few selected SNP markers are examined per patient with a low cost per assay.

Sensitive detection of point mutation using exponential strand displacement amplification-based surface enhanced Raman spectroscopy

“…So far, a variety of methods have been developed for the point mutation assay, such as ligase-mediated detection (Landegren et al, 1988;Huang et al, 2009), primer extension-based method (Sokolov, 1990;Litos et al, 2007), flap endonuclease-based cleavage method (Lyamichev et al, 1999;Chen et al, 2005) and polymerase chain reaction (PCR)-based method (Hacia et al, 1998;Fujii et al, 2000;Germer et al, 2000). Among these approaches, PCR-based method dominates the field of point mutation analysis owing to its high amplification efficiency.…”

Noble Metal Nanoparticles for Biosensing Applications

“…Finally, to detect the presence of the DNA target in the streptavidin spot, poly-dT gold nanoprobes are used to hybridize with the poly-dA probe. Other variants of this technique have also been described by Kalogianni et al to fit a specific application (e.g., SNP detection, multiplex analysis) [53–55] and by Fan et al to monitor protein-protein interactions using silver NPs instead of gold NPs [56]. Using a high-throughput microarray approach, Mirkin’s group used gold nanoprobes to substitute the conventional fluorescence-labeled probes usually used to report target hybridization [57].…”

Real-time fluorescence ligase chain reaction for sensitive detection of single nucleotide polymorphism based on fluorescence resonance energy transfer

“…The PCR amplification may introduce uncontrolled bias in the template replication, and thus, false positive signal can be observed (Harris et al, 2008). There are many detection strategies have been reported for discrimination of two alleles (Syvänen, 2001;Litos et al, 2007), including hybridization with allele-specific oligonucleotides (Saiki et al, 1989), allelespecific primer extension (Litos et al, 2007;Duan et al, 2007), minisequencing (Zhou et al, 2001), ligation of allele-specific probes (Landegren et al, 1988;Liu et al, 2013;Shi et al, 2011), and invasive cleavage with endonuclease (Lyamichev et al, 1999;Hall et al, 2000). The allele-specific PCR, by using a primer with the nucleotide at its 3′-end complementary to the nucleotide of detected target at the SNP site, can integrate the PCR amplification and allele discrimination (Duan et al, 2009;Choi et al, 2012).…”