Analytical Methods
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2012
·Vol. 5
·期 1
·285-290
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IF 1.547
Xia Liu,Tao Li,Dianjun Liu,Zhenxin Wang
Here, a three-dimensional (3D) oligonucleotide microarray-based fluorescent assay has been developed for single nucleotide polymorphisms (SNPs) and allele frequency determination. The 3D oligonucleotide microarray is fabricated on a home-made polyacrylamide hydrogel microarray which is generated by UV light photopolymerization of tiny acrylamide monomer droplets on a PEG terminated glass slide. After activation by glutaraldehyde (GA), the polyacrylamide hydrogel is able to react with NH2-modified oligonucleotides with high efficiency to generate a high signal-to-background ratio without the conventional blocking step. We demonstrate that the assay is able to detect complimentary 25 mer oligonucleotide target (fragment of a tumor suppressor gene P53) down to 100 pM and SNP with true-to-false ratios (the ratio of fluorescence signal from perfect matched DNA strands to that from the one-base mismatched DNA strands) above 20. Taking advantage of the high specificity of the assay, allele frequency as low as 2% can be accurately determined.
研究领域:
生物化学
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分子生物学
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基因组学
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DOI
高影响因子
ANALYST
·
2019
·Vol. 145
·期 1
·172-176
·
IF 4.019
Qian-Yu Zhou,Xin-Ying Zhong,Ling-Li Zhao,Li-Juan Wang,Ying-Lin Zhou,Xin-Xiang Zhang
Single nucleotide polymorphisms (SNPs) have been proven to be important biomarkers for disease diagnosis, prognosis and disease pathogenesis. Here, taking the advantages of a self-assembled oligonucleotide sandwich structure and robust chemical reactions, we have developed a simple, high-throughput and effective colorimetric analytical technique termed CuAAC-based ligation-assisted assays (CuAAC-LA) for SNP detection using a DNA-BIND 96-well plate. With the 5′-azide and 3′-alkyne groups labelled on two oligonucleotide probes, the target DNA can direct a Cu(I)-catalyzed alkyne–azide cycloaddition (CuAAC) click reaction. Since the small difference in duplex stability caused by a single-nucleotide mismatch was amplified by the steric effects of these reactive groups for the ligation reaction of an unstable duplex, CuAAC-LA exhibited an ultra-sensitive discrimination ability for a mutant type target in the presence of large amounts of wild type targets. As low as 0.05% SNP could be clearly detected, which was better than most previously reported methods by various DNA ligases, indicating that a simple and rapid synthetic method i.e., the DNA template-directed click reaction held the potential to replace the ligase for SNP detection.
研究领域:
分子生物学
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基因组学
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生物技术
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分析化学
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DOI
PubMed
高影响因子
ANALYST
·
2011
·Vol. 136
·期 21
·4480-4485
·
IF 3.913
Shujuan Xu,Yong Shao,Kun Ma,Qinghua Cui,Guiying Liu,Fei Wu,Minjie Li
DNA single-nucleotide polymorphism (SNP) detection has attracted much attention due to mutation-related diseases. Various fluorescence methods for SNP detection have been proposed and many are already in use. However, fluorescence enhancement for signal-on SNP identification without label modification still remains a challenge. Here, we find that the abasic site (AP site) in a DNA duplex can be developed as a binding pocket favorable for the occurrence of the excited-state intramolecular proton transfer (ESIPT) of a 3-hydroxyflavone, fisetin, which is used as a proof of concept for effective SNP identification. Fisetin binding at the AP site is highly selective for target thymine or cytosine facing the AP site by observation of a drastic increase in the ESIPT emission band. In addition, the target recognition selectivity based on this ESIPT process is not affected by flanking bases of the AP site. The binding selectivity of fisetin at the AP site is also confirmed by measurements of fluorescence resonance energy transfer, emission lifetime and DNA melting. The fluorescent signal-on sensing for SNP based on this fluorophore is substantially advantageous over the previously used fluorophores such as the AP site-specific signal-off organic ligands with a similar fluorescing mechanism before and after binding to DNA with hydrogen bonding interaction. We expect that this approach will be employed to develop a practical SNP detection method by locating an AP site toward a target and employing an ESIPT probe as readout.
研究领域:
化学
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生物化学
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分子生物学
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遗传学
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2020
·
IF 5.977
Fang Hu,Wancun Zhang,Wei Meng,Yuxiang Ma,Xianwei Zhang,Ying Xu,Peng Wang,Yueqing Gu
Single nucleotide polymorphisms (SNPs) are crucial during the early diagnosis of a given disease as well as in evaluating their response to certain drugs. Thus, this study sought the development of ferrocene (Fc)-labeled electrochemical biosensor for SNP detection. This proposed system involves the ligation of four short probes (e.g., A, B, A′, and B′, where B′ is labeled with an Fc-tag) in the presence of target DNA via ligase chain reaction (LCR), resulting in the formation of Fc-tagged duplex AB-A′B′ in 2 n . Subsequently, immobilization of the Fc-tagged duplex AB-A′B′ on a single-stranded DNA capture probe (SC-DNA)-carboxyl multi-wall carbon nanotube (MWCNT-COOH) modified glassy carbon electrode (GCE) was accomplished through hybridization. Owing to the specificity of hybridization, and the use of Fc as electrochemical probe for detection of duplex AB-A′B′, such strategy realized directly analysis of LCR products without the need for purification . By taking advantage of the thermal stability and high-discrimination ability of HiFi Taq DNA ligase for single-base differences, the specificity of hybridization, the EGFR T790 M mutant DNA (MT-DNA) biosensor was developed to offer a low limit of detection (0.75 aM), a high discrimination of single-base mismatches [as low as 0.01% (molar fraction)], a wide linear range of more than 7 orders of magnitude (1 aM–10 pM), and the recovery rates (95.3%–107.8%) from human serum samples. Thus, the biosensor under development was found to be economical, highly-sensitive, and exceptionally selective for detection of SNPs, and as well as extending the versatile applications of LCR to offer great potential for diagnosis and individual clinical regimens.
研究领域:
生物化学
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生物技术
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分析化学
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分子生物学
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医学诊断
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DOI
PubMed
高影响因子
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