Guohua Zhou

Nanjing University, Nan-ching, Jiangsu Sheng, China

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Publications (52)176.96 Total impact

  • Yunlong Liu · Bingjie Zou · Haiping Wu · Yanan Chu · Guohua Zhou
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    ABSTRACT: Invader Plus, which combines PCR amplification with invasive cleavage-based signal amplification, is an efficient method for genotyping. However, the non-specific signals in Invader assay are caused by the hybridization of the wild-type probe (target) with the mutated target (probe), leading to a false-positive typing result. To increase the specificity of Invader Plus assays, we proposed to introduce an artificially mismatched base into the region next to the invasive site of the probe. The mismatched base efficiently reduced the thermostability of non-specific invasive structures, therefore the non-specific signals decreased dramatically. We investigated various positions for introducing the mismatched base, and found that the best position is the nucleotide right next to the invasive site. We next genotyped the aldehyde dehydrogenase-2 gene polymorphisms which are related to the individualized medicine of nitroglycerin and the risk of esophageal cancers. The results showed that the non-specific signals from the wild-type probe in the mutated target were significantly reduced by using the mismatched probe. Our improved-Invader Plus method can achieve more accurate genotyping in comparison with the conventional Invader Plus assay, which is more feasible for clinical genotyping tests.
    No preview · Article · Dec 2015 · Analytical methods
  • Shuo Li · Lizhou Sun · Guohua Zhou · Huan Huang
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    ABSTRACT: Early detection of abnormal expression levels of cancer-related genes is crucial for reducing cancer mortality. Here, we describe a dye-free multiplex bioassay for simultaneous and quantitative analysis of the expression levels of multiple genes from one sample in a single assay, based on Sequence-tagged Multiplex ligationdependent probe Amplification (MLPA) coupled with pyrosequencing (termed as “SMAP”). Each pair of MLPA probes, containing a designed barcode, represents a gene of interest; thus, the use of various dyes to label different genes was avoided. The unique three-base barcode design on the probes, which can be decoded by pyrosequencing, enables individual quantification of the expression levels of six genes. Moreover, a new carryover contamination prevention system based on the use of restriction endonucleases was developed for PCR-based diagnostic screening assays. SMAP analysis revealed significant differences between the expression levels of CRC-related genes in the tumor tissues and normal tissues from a CRC patient. For PCR-based diagnostic screening assays, 0.5 U of the FokI restriction endonuclease was sufficient for the removal 0.01 pmol of PCR contamination. The ability to analyze the expression levels of a greater number of cancer-related genes would improve diagnostic sensitivity and efficacy. SMAP is amenable to the detection of an increased number of genes by lengthening the artificially designed barcodes; thus our method provides a promising means for cancer diagnostics and improving the treatment options available to cancer patients. © 2015, The Korean Society for Biotechnology and Bioengineering and Springer-Verlag Berlin Heidelberg.
    No preview · Article · Nov 2015 · Biotechnology and Bioprocess Engineering
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    ABSTRACT: Genetic polymorphism and environment each influence individual variability in drug metabolism and disposition. It is preferable to predict such variability, which may affect drug efficacy and toxicity, before drug administration. We examined individual differences in the pharmacokinetics of atorvastatin by applying gas chromatography-mass spectroscopy (GC-MS)-based metabolic profiling to pre-dose plasma samples from 48 healthy volunteers. We determined the level of atorvastatin in plasma using LC/MSMS. With the endogenous molecules which showed a good correlation with pharmacokinetic parameters, a refined partial least squares model was calculated based on pre-dose data from a training set of 36 individuals, and exhibited good predictive capability for the other 12 individuals in the prediction set. In addition, the model was successfully used to predictively classify individual pharmacokinetic responses into subgroups. Metabolites such as tryptophan, alanine, arachidonic acid, 2-Hydroxybutyric acid, cholesterol and isoleucine were indicated as candidate markers for predicting, showing better predictive capability for explaining individual differences than conventional physiological index. These results suggest that a pharmacometabonomic approach offers the potential to predict individual differences in pharmacokinetics, and therefore to facilitate individualized drug therapy.
    No preview · Article · Jul 2015 · Journal of Proteome Research
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    ABSTRACT: One of the major reasons for pregnant women to ask for prenatal diagnosis is to detect fetal chromosomal aneuploidies. Analysis of allele ratios of SNPs has been used for prenatal detection of fetal aneuploidies using MALDI-TOF mass spectrometry (MS). However, quantitative SNP genotyping by MALDI-TOF MS is challenging. To obtain a better quantification of allelic ratios, a Pyrosequencing(®) protocol for SNP genotyping has been developed to perform prenatal diagnosis of aneuploidies.To avoid the laborious process and risk of cross-contamination brought in by DNA extraction procedures, a PCR assay, which can amplify DNA directly from cells in amniotic fluid, has been developed. Pre-amplification steps such as cell enrichment and heating are required to obtain sufficient amounts of amplification products.In this chapter, SNPs on chromosome 21 are used to detect trisomy 21 as an example of aneuploidy by quantifying the allele ratio using Pyrosequencing. Primer selection for PCRs and Pyrosequencing reactions, optimization of nucleotide dispensation orders, establishment of cutoff values for trisomy 21, and interpretation of data are all factors essential for a successful diagnosis and are discussed in detail herein.
    No preview · Article · Jun 2015 · Methods in molecular biology (Clifton, N.J.)
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    Huan Huang · Shuo Li · Lizhou Sun · Guohua Zhou
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    ABSTRACT: To simultaneously analyze mutations and expression levels of multiple genes on one detection platform, we proposed a method termed "multiplex ligation-dependent probe amplification-digital amplification coupled with hydrogel bead-array" (MLPA-DABA) and applied it to diagnose colorectal cancer (CRC). CRC cells and tissues were sampled to extract nucleic acid, perform MLPA with sequence-tagged probes, perform digital emulsion polymerase chain reaction (PCR), and produce a hydrogel bead-array to immobilize beads and form a single bead layer on the array. After hybridization with fluorescent probes, the number of colored beads, which reflects the abundance of expressed genes and the mutation rate, was counted for diagnosis. Only red or green beads occurred on the chips in the mixed samples, indicating the success of single-molecule PCR. When a one-source sample was analyzed using mixed MLPA probes, beads of only one color occurred, suggesting the high specificity of the method in analyzing CRC mutation and gene expression. In gene expression analysis of a CRC tissue from one CRC patient, the mutant percentage was 3.1%, and the expression levels of CRC-related genes were much higher than those of normal tissue. The highly sensitive MLPA-DABA succeeds in the relative quantification of mutations and gene expressions of exfoliated cells in stool samples of CRC patients on the same chip platform. MLPA-DABA coupled with hydrogel bead-array is a promising method in the non-invasive diagnosis of CRC.
    Preview · Article · Apr 2015 · PLoS ONE
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    ABSTRACT: Colorimetric DNA detection is preferable to methods in clinical molecular diagnostics, because no expensive equipment is required. Although many gold nanoparticle-based colorimetric DNA detection strategies have been developed to analyze DNA sequences of interest, few of them can detect somatic mutations due to their insufficient specificity. In this study, we proposed a colorimetric DNA detection method by coupling invasive reaction with nicking endonuclease-assisted nanoparticles amplification (IR-NEANA). A target DNA firstly produces many flaps by invasive reaction. Then the flaps are converted to targets of nicking reaction-assisted nanoparticles amplification by ligation reaction to produce the color change of AuNPs, which can be observed by naked eyes. The detection limit of IR-NEANA was determined as 1 pM. Most importantly, the specificity of the method is high enough to pick up as low as 1% mutant from a large amount of wild-type DNA backgrounds. The EGFR gene mutated at c.2573 T>G in 9 tissue samples from non-small cell lung cancer patients were successfully detected by using IR-NEANA, suggesting that our proposed method can be used to detect somatic mutations in biological samples.
    No preview · Article · Apr 2015 · Biosensors & Bioelectronics
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    ABSTRACT: The high background signal caused by non-specific amplification (NSA) is a serious issue when using the conventional exponential amplification reaction (EXPAR). We describe a novel method of suppressing NSA using 10 mg L−1 graphene oxide (GO) to prevent non-specific binding of DNA polymerase to the template, resulting in ultra-low background. A side effect of the use of GO is the slow release of ssDNA from the GO surface, with the positive signal decreasing accordingly. The problem of low signal intensity is addressed by applying a 2 μM solution of single-stranded binding protein (SSB) to accelerate the release of template for EXPAR. The use of GO and SSB in EXPAR can substantially suppress NSA, but it does not compromise the performance of the quantification step. The improved EXPAR presented here can detect concentration as low as 5 aM of trigger, which is approximately four orders of magnitude lower than that of conventional EXPAR under the same experimental conditions. Graphical Abstract A high background in exponential amplification reaction (EXPAR) is effectively suppressed by using graphene oxide (GO). The slow release of ssDNA from GO surface is countered by using single-stranded binding protein. This improved EXPAR can detect as little as 5 aM of a trigger.
    Full-text · Article · Dec 2014 · Microchimica Acta
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    ABSTRACT: A novel DNA detection assay was proposed by invasive reaction coupled with molecular beacon assisted strand-displacement signal amplification (IRASA). Target DNAs are firstly hybridized to two probes to initiate invasive reaction to produce amplified flaps. Then these flaps are further amplified by strand-displacement signal amplification.The detection limit was around 0.2 pM.
    No preview · Article · Sep 2014 · Chemical Communications
  • Qinxin Song · Guijiang Wei · Guohua Zhou
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    ABSTRACT: A portable bioluminescence analyser for detecting the DNA sequence of genetically modified organisms (GMOs) was developed by using a photodiode (PD) array. Pyrosequencing on eight genes (zSSIIb, Bt11 and Bt176 gene of genetically modified maize; Lectin, 35S-CTP4, CP4EPSPS, CaMV35S promoter and NOS terminator of the genetically modified Roundup ready soya) was successfully detected with this instrument. The corresponding limit of detection (LOD) was 0.01% with 35 PCR cycles. The maize and soya available from three different provenances in China were detected. The results indicate that pyrosequencing using the small size of the detector is a simple, inexpensive, and reliable way in a farm/field test of GMO analysis.
    No preview · Article · Jul 2014 · Food Chemistry
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    ABSTRACT: Pyrosequencing is a powerful tool widely used in genetic analysis, however template preparation prior to pyrosequencing is still costly and time-consuming. To achieve an inexpensive and labor-saving template preparation for pyrosequencing, we have successfully developed a single-tube multiplex PCR including a pre-amplification and a universal amplification. In the process of pre-amplification, a low concentration of target-specific primers tagged with universal ends introduced universal priming regions into amplicons. In the process of universal amplification, a high concentration of universal primers was used for yielding amplicons with various SNPs of interest. As only a universal biotinylated primer and one step of single-stranded DNA preparation were required for typing multiple SNPs located on different sequences, pyrosequencing-based genotyping became time-saving, labor-saving, sample-saving, and cost-saving. By a simple optimization of multiplex PCR condition, only a 4-plex and a 3-plex PCR were required for typing 7 SNPs related to tamoxifen metabolism. Further study showed that pyrosequencing coupled with an improved multiplex PCR protocol allowed around 30% decrease of either typing cost or typing labor. Considering the biotinylated primer and the optimized condition of the multiplex PCR are independent of SNP locus, it is easy to use the same condition and the identical biotinylated primer for typing other SNPs. The preliminary typing results of the 7 SNPs in 11 samples demonstrated that multiplex PCR-based pyrosequencing could be promising in personalized medicine at a low cost.
    No preview · Article · Jun 2014 · Journal of Nanoscience and Nanotechnology
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    ABSTRACT: The aim of this study was to evaluate (+)-catechin and (-)-epigallocatechin gallate (EGCG) cellular uptake and transport across human intestinal Caco-2 cell monolayer in both the absence and presence of niosomal carrier in variable conditions. The effect of free drugs and drug-loaded niosomes on the growth of Caco-2 cells was studied. The effects of time, temperature, and concentration on drug cellular uptake in the absence or presence of its niosomal delivery systems were investigated. The intestinal epithelial membrane transport of the drug-loaded niosomes was examined using the monolayer of the human Caco-2 cells. The kinetics of transport, and the effect of temperature, adenosine triphosphate inhibitor, permeability glycoprotein inhibitor, multidrug resistance-associated protein 2 inhibitor, and the absorption enhancer on transport mechanism were investigated. It was found that the uptake of catechin, EGCG, and their niosomes by Caco-2 cells was 1.22±0.16, 0.90±0.14, 3.25±0.37, and 1.92±0.22 μg/mg protein, respectively (n=3). The apparent permeability coefficient values of catechin, EGCG, and their niosomes were 1.68±0.16, 0.88±0.09, 2.39±0.31, and 1.42±0.24 cm/second (n=3) at 37°C, respectively. The transport was temperature- and energy-dependent. The inhibitors of permeability glycoprotein and multidrug resistance-associated protein 2 and the absorption enhancer significantly enhanced the uptake amount. Compared with the free drugs, niosomal formulation significantly enhanced drug absorption. Additionally, drug-loaded niosomes exhibited stronger stability and lower toxicity. These findings showed that the oral absorption of tea flavonoids could be improved by using the novel drug delivery systems.
    Preview · Article · May 2014 · International Journal of Nanomedicine
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    ABSTRACT: Pyrosequencing has been one of the most commonly used methods for genotyping; however, generally it needs single-stranded DNA (ssDNA) preparation from PCR amplicons as well as purified genomic DNA extraction from whole blood. To simplify the process of a pyrosequencing protocol, we proposed an improved linear-after-the-exponential (LATE)-PCR by employing whole blood as the starting material. A successful LATE-PCR was achieved by using a common Taq DNA polymerase in high pH buffer (HpH-buffer). As amplicons from LATE-PCR contain a large amount of ssDNA, pyrosequencing can be performed on the amplicons directly. Since DNA extraction and ssDNA preparation are omitted, the labor, cost and cross-contamination risk is decreased compared to conventional pyrosequencing-based genotyping protocols. The results for typing three polymorphisms related to personalized medicine of fluorouracil indicate that the proposed whole-blood LATE-PCR can be well coupled with pyrosequencing, thus becoming a potential tool in personalized medicine.
    No preview · Article · Mar 2014 · Analytical methods
  • Bingjie Zou · Yinjiao Ma · Guohua Zhou
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    ABSTRACT: Although many approaches based on template replication were developed and applied in DNA detection, cross-contamination from amplicons is always a vexing problem. Thus, signal amplification is preferable for DNA detection due to its low risk of cross-contamination from amplicons. Here, we proposed a cascade enzymatic signal amplification (termed as CESA) by coupling Afu flap endonuclease with nicking endonuclease, including three steps: invasive signal amplification, flap ligation, and nicking endonuclease signal amplification. Because of the advantages of low risk of contamination, no sequence requirement of target DNA, and the universal reaction conditions for any target detection, CESA has a great potential in clinical diagnosis.
    No preview · Article · Sep 2013 · Methods in molecular biology (Clifton, N.J.)
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    ABSTRACT: 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).
    No preview · Article · Jul 2013 · The Analyst
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    ABSTRACT: A novel assay based on a solvent for inducing the aggregation of AuNPs colloid was proposed to discriminate ssDNA from dsDNA. Eleven organic solvents with different polarity were investigated, and it was found that DMSO was possible to aggregate AuNPs at the amount of only 0.4 microL in a 50-microL detection system. Further research showed that 0.8 microL of DMSO could discriminate the ssDNA from dsDNA. Colorimetric detection with various conditions, including the ratio of the target to the probe, and the concentration of AuNPs and DNA, was investigated. The proposed method was successfully used for SNP typing, and unambiguous discrimination of a wild type from a mutant was obtained for the templates with the mismatched base at the 5'-end or in the middle of the target sequence. As no requirement of gold modification and detection instrument, we believe that this method will be much low in the cost for DNA detection.
    No preview · Article · Jun 2013 · Journal of Nanoscience and Nanotechnology
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    ABSTRACT: LAMP is an isothermal amplification method that can achieve ultra-high sensitivity and specificity. However, the conventional detection of LAMP amplicons can lead to cross-contamination due to the need to open the reaction tube which contains a large number of amplicons. To achieve closed-tube LAMP detection, we have developed a method that separates a solution of SYBR Green I (SGI) from the LAMP reagents using temperature-sensitive wax. The SGI is sealed in the bottom of the tube so not to interfere with the LAMP reaction, but is released into the mixture after the completion of the reaction by melting the wax. To enable the analysis of the closed-tube LAMP samples automatically, an instrument based on this new method was constructed. The background measurement of the LAMP due to primer dimers was significantly reduced by detecting the amplicons at 75 degrees C. HBV and 2009 H1N1 virus were successfully analyzed by the LAMP assay using tubes containing wax-sealed SGI and the prototype instrument, indicating that the method has the advantage of easy set-up (no extra components need to be added into the LAMP mixture for detection), high sensitivity (fluorescent intercalator), low background (detected at 75 degrees C) and no cross-contamination (closed-tube). Therefore, the novel LAMP detection, coupled with the instrument has the potential to be a diagnostic tool for a number of clinical applications in hospitals as well as on-site screening of pathogenic agents.
    No preview · Article · Jun 2013 · Journal of Nanoscience and Nanotechnology
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    ABSTRACT: Allelic ratio of an SNP has been used for prenatal diagnosis of fetal trisomy 21 by MALDI-TOF mass spectrometry (MS). Because MALDI-TOF MS is challenging in quantification performance, pyrosequencing was proposed to replace MS by better quantification of allelic ratios. To achieve a simple and rapid clinical diagnostic, PCR with "HpH Buffer" (a buffer with a high pH) was developed to directly amplify amniotic fluid. By the established assay, 114 samples of amniotic fluid were analyzed by pyrosequencing five SNPs of each sample; the allelic ratios of euploid heterozygotes were thus calculated to determine the cut-off values for prenatal diagnosis of trisomy 21. The panel of five SNPs were high in heterozygosity so that at least one heterozygote was found in each sample, and 86% of the samples had at least two heterozygotes, giving a nearly 100% sensitivity (population coverage) of the assay. By using the cut-off values of each SNP, 20 pre-diagnosed clinical samples were detected as trisomy 21 carriers with a confidence level over 99%, indicating that our method and karyotyping analysis were consistent in results. In conclusion, this pyrosequencing-based approach, coupled with direct amplification of amniotic fluid, is accurate in quantitative genotyping and simple in operation. We believe that the approach could be a promising alternative to karyotyping analysis in prenatal diagnosis.
    No preview · Article · Mar 2013 · The Analyst
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    ABSTRACT: Quantitative analysis of virus nucleic acids is essential for monitoring the efficacy of medical treatment based on the copy numbers of virus's RNA or DNA in blood. To quantitatively detect virus nucleic acids in blood, here an internal amplification control (IAC) coupled with a nanoparticle-based DNA biosensor was proposed. The IACs with a specific sequence were designed and spiked into serum before nucleic acids extraction. Sequences of the IACs and the targets only differ in the base order of one PCR priming site; thus, the IACs and the targets are identical in Tm, giving the same amplification efficiency during PCR. To visually detect amplicons, a dipstick biosensor based on streptavidin-functionalized nanoparticles is employed. By comparing color densities of a test zone with an IAC zone on the biosensor, the content of the target in serum can be semi-quantitatively analyzed. This approach has achieved the detection of HBV DNA at approximately 100 copies of the pathogen load. The feasibility of this method is demonstrated by successful semi-quantification of pathogen load in 30 clinical samples from HBV-infected patients. These data indicate that the introduction of an IAC and nanoparticle-based dipstick-type biosensor could be a powerful tool in point of care testing (POCT).
    No preview · Article · Nov 2012 · Biosensors & Bioelectronics
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    ABSTRACT: A 3-dimensional (3-D) polyacrylamide gel microarray based on dual-color fluorescence hybridization was an efficient SNP typing method with a high-throughput, but it is expensive to use dual dye-labeled allele-specific probes to type various SNPs. To lower the typing cost on 3-D polyacrylamide gel microarray, we propose a novel method by incorporating Cy5-dCTP into label-free allele-specific probes hybridizing to gel-immobilized targets. The method is much simple. At first, raw PCR products without any purification was spotted on the acryl-modified slides to copolymerize with acrylamide monomers. Then a pair of allele-specific probes were respectively added into two different areas of a hydrogel chip to hybridize with the single-stranded DNA targets immobilized in the gel-pads. Before extension reaction with Cy5-dCTP, electrophoresis was performed on the gel chip to remove non-specific allele-specific probes, and a high specificity was thus obtained. After the extension reaction, electrophoresis was used once more to remove the unincorporated Cy5-dCTP absorbed in the gel pads, and a low background image was achieved. The method was successfully employed to type the SNP (C14417G) in the OLR-1 gene for 40 different samples, and the typing results were consistent with those by pyrosequencing, indicating that the proposed method is accurate and specific in SNP typing. As no use of dye-modified probes, the typing cost is significantly decreased in comparison with the conventional typing method based on dual-color fluorescence hybridization, in particular when typing multiple SNPs. In addition to the low cost, our method has a low risk of cross-contamination from PCR amplicons due to no need of purification step of PCR products. Although only proof-of-concept results were given, we believe that the proposed method should be very useful for screening the biomarkers related to disease-susceptibility and personalized medicine where detection of many SNPs in different genes is required.
    No preview · Article · Sep 2012 · Journal of Nanoscience and Nanotechnology
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    ABSTRACT: Somatic mutations in stool DNA are quite specific to colorectal cancer (CRC), but a method being able to detect the extraordinarily low amounts of mutants is challengeable in sensitivity. We proposed a hydrogel bead-array to digitally count CRC-specific mutants in stool at a low cost. At first, multiplex amplification of targets containing multiple mutation loci of interest is carried out by a target enriched multiplex PCR (Tem-PCR), yielding the templates qualified for emulsion PCR (emPCR). Then, after immobilizing the beads from emPCR on a glass surface, the incorporation of Cy3-dUTP into the mutant-specific probes, which are specifically hybridized with the amplified beads from emPCR, is used to color the beads coated with mutants. As all amplified beads are hybridized with the Cy5-labeled universal probe, a mutation rate is readily obtained by digitally counting the beads with different colors (yellow and red). A high specificity of the method is achieved by removing the mismatched probes in a bead-array with electrophoresis. The approach has been used to simultaneously detect 8 mutation loci within the APC, TP53, and KRAS genes in stools from eight CRC patients, and 50% of CRC patients were positively diagnosed; therefore, our method can be a potential tool for the noninvasive diagnosis of CRC.
    No preview · Article · Jun 2012 · Analytical Chemistry

Publication Stats

358 Citations
176.96 Total Impact Points

Institutions

  • 2006-2015
    • Nanjing University
      • • School of Medicine
      • • Department of Chemical Engineering
      Nan-ching, Jiangsu Sheng, China
  • 2006-2014
    • China Pharmaceutical University
      • • School of Pharmacy
      • • School of Life Science and Technology
      • • Division of Analytical Chemistry
      Nan-ching-hsü, Jiangxi Sheng, China
  • 2001
    • Daiwa House Central Research Laboratory
      Edo, Tōkyō, Japan