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Yang B.,Nanjing University | Zhou G.,Nanjing University | Zhou G.,Huadong Research Institute for Medicine and Biotechnics | Huang L.L.,Nanjing University
Analytical and Bioanalytical Chemistry | Year: 2010

Single nucleotide polymorphisms (SNPs) represent the most abundant source of genetic variation in the human genome, and they can be linked to genetic susceptibilities or varied pharmaceutical responses. Established SNP detection techniques are mainly PCR-based, which means that they involve complex, labor-intensive procedures, are easy contaminated, and can give false-positive results. Therefore, we have developed a simple and rapid MS-based disulfide barcode methodology that relies on magnifying the signal from a dual-modified gold nanoparticle. This approach permits direct SNP genotyping of total human genomic DNA without the need for primer-mediated enzymatic amplification. Disulfides that are attached to the gold nanoparticle serve as a "barcode" that allows different sequences to be discerned using MS detection. Specificity is based on two sequential oligonucleotide hybridizations, which include two steps: the first is the capture of the target by gene-specific probes immobilized onto magnetic beads; the second is the recognition of gold nanoparticles functionalized with allele-specific oligonucleotides. The sensitivity of this new method reaches down to the 0.1 fM range, thus approaching that of PCR. The feasability of this SNP identification methodology based on an MS-based disulfide barcode assay was demonstrated by applying it to genomic DNA samples representing all possible genotypes of the SNPs G2677T and C3435T in the human MDR1 gene. Due to its great advantage-the ability to perform SNP typing without the use of PCR-the assay was found to be simple, rapid and robust, and so may be highly suited to routine clinical detection as well as basic medical research. © 2010 Springer-Verlag. Source


Zou B.,Huadong Research Institute for Medicine and Biotechnics | Zou B.,China Pharmaceutical University | Ma Y.,Huadong Research Institute for Medicine and Biotechnics | Ma Y.,China Pharmaceutical University | And 5 more authors.
Angewandte Chemie - International Edition | Year: 2011

Hail CESA! Cascade enzymatic signal amplification (CESA) for DNA detection is achieved in three steps: invasive signal amplification by Afu endonuclease to generate amplified flaps, flap ligation by T4 ligase to form a nicking site, and nicking reaction by nicking endonuclease to produce amplified signals (see picture for last step). The sensitivity of CESA is as high as 1 fM DNA. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source


Zou B.,China Pharmaceutical University | Ma Y.,China Pharmaceutical University | Wu H.,Huadong Research Institute for Medicine and Biotechnics | Zhou G.,China Pharmaceutical University | And 2 more authors.
Analyst | Year: 2012

Detection of nucleic acids with signal amplification is preferable in clinical diagnosis. A novel approach was developed for signal amplification by coupling invasive reaction with hyperbranched rolling circle amplification (HRCA). Invasive reaction, which does not rely on specific recognition sequences in a target but a specific structure formed by the specific binding of an upstream probe and a downstream probe to a target DNA, can generate thousands of flaps from one target DNA; then the flaps are ligated with padlock probes to form circles, which are the templates of HRCA. As HRCA amplicon sequence is free of target DNA sequence, signal amplification is achieved. Because flap sequence is the same to any target of interest, HRCA is universal; the detection cost is hence greatly reduced. The sensitivity of the proposed method is less than 1 fM artificial DNA targets; and the specificity of the method is high enough to discriminate one base difference in the target sequence. The feasibility was verified by detecting real biological samples from HBV carriers, indicating that the method is highly sensitive, cost-effective, and has a low risk of cross-contamination from amplicons. These properties should give great potential in clinical diagnosis. © 2012 The Royal Society of Chemistry. Source


Song Q.,China Pharmaceutical University | Wu H.,China Pharmaceutical University | Wu H.,Huadong Research Institute for Medicine and Biotechnics | Feng F.,China Pharmaceutical University | And 5 more authors.
Analytical Chemistry | Year: 2010

Although the pyrosequencing method is simple and fast, the step of ssDNA preparation increases the cost, labor, and cross-contamination risk. In this paper, we proposed a method enabling pyrosequencing directly on dsDNA digested by nicking endonucleases (NEases). Recognition sequence of NEases was introduced using artificially mismatched bases in a PCR primer (in the case of genotyping) or a reverse-transcription primer (in the case of gene expression analysis). PCR products were treated to remove excess amounts of primers, nucleotides, and pyrophosphate (PPi) prior to sequencing. After the nicking reaction, pyrosequencing starts at the nicked 3' end, and extension reaction occurs when the added dNTP is complementary to the non-nicked strand. Although the activity of strand displacement by Klenow is limited, ∼10 bases are accurately sequenced; this length is long enough for genotyping and SRPP-based differential gene expression analysis. It was observed that the signals of two allelespecific bases in a pyrogram from nicked dsDNA are highly quantitative, enabling quantitative determination of allele-specific templates; thus, Down's Syndrome diagnosis as well as differential gene expression analysis was successfully executed. The results indicate that pyrosequencing using nicked dsDNA as templates is a simple, inexpensive, and reliable way in either quantitative genotyping or gene expression analysis. © 2010 American Chemical Society. Source


Song Q.,China Pharmaceutical University | Yang H.,Huadong Research Institute for Medicine and Biotechnics | Zou B.,Nanjing University | Kajiyama T.,Hitachi Ltd. | And 3 more authors.
Analyst | Year: 2013

Nucleic acid analysis in a single cell is very important, but the extremely small amount of template in a single cell requires a detection method more sensitive than the conventional method. In this paper, we describe a novel assay allowing a single cell genotyping by coupling improved linear-after-the- exponential-PCR (imLATE-PCR) on a modified glass slide with highly sensitive pyrosequencing. Due to the significantly increased yield of ssDNA in imLATE-PCR amplicons, it is possible to employ pyrosequencing to sequence the products from 1 μL chip PCR which directly used a single cell as the starting material. As a proof-of-concept, the 1555A>G mutation (related to inherited deafness) on mitochondrial DNA and the SNP 2731C>T of the BRCA1 gene on genomic DNA from a single cell were successfully detected, indicating that our single-cell-pyrosequencing method has high sensitivity, simple operation and is low cost. The approach has promise to be of efficient usage in the fields of diagnosis of genetic disease from a single cell, for example, preimplantation genetic diagnosis (PGD). This journal is © The Royal Society of Chemistry. Source

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