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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.
The Analyst 03/2013; · 4.23 Impact Factor
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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).
Biosensors & bioelectronics 11/2012; 42C:261-266. · 5.43 Impact Factor
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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.
Journal of Nanoscience and Nanotechnology 09/2012; 12(9):6887-92. · 1.56 Impact Factor
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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.
Analytical Chemistry 06/2012; 84(13):5645-52. · 5.86 Impact Factor
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ABSTRACT: Pyrosequencing is a tool based on bioluminescence reaction for real-time analyzing DNA sequences. The sensitivity of pyrosequencing mainly depends on luciferase in reaction mixture. However, the instability of pyrosequencing reagents caused by fragile wild Photinus pyralis luciferase (PpL) in conventional pyrosequencing usually leads to unsatisfied results, which limits the application of pyrosequencing. In order to improve the stability of pyrosequencing reagents, the coding sequences of mutant thermostable Luciola lateralis luciferase (rt-LlL) was synthesized, and inserted into the plasmid of pET28a(+) to express the thermostable rt-LlL with a 6 x His-tag in the N terminal. The purified rt-LlL with the molecular mass of 60 kDa was obtained by Ni-affinity chromatography. The specific activity of rt-LlL was determined as 4.29 x 10(10) RLU/mg. Moreover, the thermostability of rt-LlL was investigated, and the results showed that rt-LlL had activity at 50 degrees C, and remained 90% of activity after incubated at 40 degrees C for 25 min. Finally, rt-LlL was used to substitute commercial Photinus pyralis luciferase in conventional pyrosequencing reagent to get thermostable pyrosequencing reagent. Comparing with conventional pyrosequencing reagent, the thermostable pyrosequencing reagent is more stable, and it's activity would not lose when incubated at 37 degrees C for 1 h. This study laid foundation of establishing reliable and stable pyrosequencing system which would be applied in Point-of-Care Testing.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 06/2012; 28(6):763-71.
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ABSTRACT: The loop-mediated isothermal amplification (LAMP) is a well-developed method for replicating a targeted DNA sequence with a high specificity, but multiplex LAMP detection is difficult because LAMP amplicons are very complicated in structure. To allow simultaneous detection of multiple LAMP products, a series of target-specific barcodes were designed and tagged in LAMP amplicons by FIP primers. The targeted barcodes were decoded by pyrosequencing on nicked LAMP amplicons. To enable the nicking reaction to occur just near the barcode regions, the recognition sequence of the nicking endonuclease (NEase) was also introduced into the FIP primer. After the nicking reaction, pyrosequencing started at the nicked 3' end when the added deoxyribonucleoside triphosphate (dNTP) was complementary to the non-nicked strand. To efficiently encode multiple targets, the barcodes were designed with a reporter base and two stuffer bases, so that the decoding of a target-specific barcode only required a single peak in a pyrogram. We have successfully detected the four kinds of pathogens including hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and Treponema pallidum (TP), which are easily infected in blood, by a 4-plex LAMP in a single tube, indicating that barcoded LAMP coupled with NEase-mediated pyrosequencing is a simple, rapid, and reliable way in multiple target identification.
Analytical Chemistry 03/2012; 84(8):3758-63. · 5.86 Impact Factor
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Zhiyao Chen,
Xiaoying Fu,
Xiaodan Zhang,
Xiqun Liu,
Bingjie Zou,
Haiping Wu,
Qinxin Song,
Jinheng Li,
Tomoharu Kajiyama,
Hideki Kambara, Guohua Zhou
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ABSTRACT: A novel dye-free labeling method for a multiplex bioassay was proposed by using short sequence-based barcodes consisting of a reporter base and repeats of two stuffer bases; then, the barcodes were quantitatively decoded by a single pyrosequencing assay without any pre-separation.
Chemical Communications 02/2012; 48(18):2445-7. · 6.17 Impact Factor
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ABSTRACT: Pyrosequencing is an ideal tool for verifying the sequence of amplicons. To enable pyrosequencing on amplicons from nucleic acid sequence-based amplification (NASBA), asymmetric NASBA with unequal concentrations of T7 promoter primer and reverse transcription primer was proposed. By optimizing the ratio of two primers and the concentration of dNTPs and NTPs, the amount of single-stranded cDNA in the amplicons from asymmetric NASBA was found increased 12 times more than the conventional NASBA through the real-time detection of a molecular beacon specific to cDNA of interest. More than 20 bases have been successfully detected by pyrosequencing on amplicons from asymmetric NASBA using Human parainfluenza virus (HPIV) as an amplification template. The primary results indicate that the combination of NASBA with a pyrosequencing system is practical, and should open a new field in clinical diagnosis.
The Analyst 12/2011; 136(24):5229-33. · 4.23 Impact Factor
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ABSTRACT: 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.
The Analyst 12/2011; 137(3):729-34. · 4.23 Impact Factor
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ABSTRACT: We expressed recombinant single-stranded DNA-binding protein (r-SSBP) from Escherichia coli with the molecular weight of 24-kDa by using genetic engineering strategy, and demonstrated the single-stranded DNA (ssDNA)-binding activity of r-SSBP by electrophoretic mobility shift assay (EMSA). To further characterize r-SSBP, we studied the effects of r-SSBP on melting temperature (T(m)) of DNA. The results showed that r-SSBP could bind to ssDNA, and lower the T(m) of DNA, especially for single-base mismatched DNA. Therefore, r-SSBP significantly increased the T(m) difference between single-base mismatched DNA and perfect matched DNA. These results are very beneficial for single-nucleotide polymorphism detection. Moreover, we applied r-SSBP in high sensitive pyrosequencing system developed by our group. The results suggest that the r-SSBP decreased non-specific signals, corrected the proportion of signal peak height and improved the performance of pyrosequencing.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 10/2011; 27(10):1513-20.
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ABSTRACT: To digitally analyze expression levels of multiple genes in one reaction, we proposed a method termed as 'MDHB' (Multiplexed Digital-PCR coupled with Hydrogel Bead-array). The template for bead-based emulsion PCR (emPCR) was prepared by reverse transcription using sequence-tagged primers. The beads recovered from emPCR were immobilized with hydrogel to form a single-bead layer on a chip, and then decoded by gene-specific probe hybridization and Cy3-dUTP based primer extension reaction. The specificity of probe hybridization was improved by using electrophoresis to remove mismatched probes on the bead's surface. The number of positive beads reflects the abundance of expressed genes; the expression levels of target genes were normalized to a housekeeping gene and expressed as the number ratio of green beads to red beads. The discrimination limit of MDHB is 0.1% (i.e., one target molecule from 1000 background molecules), and the sensitivity of the method is below 100 cells when using the β-actin gene as the detection target. We have successfully employed MDHB to detect the relative expression levels of four colorectal cancer (CRC)-related genes (c-myc, COX-2, MMP7, and DPEP1) in 8 tissue samples and 9 stool samples from CRC patients, giving the detection rates of 100% and 77%, respectively. The results suggest that MDHB could be a potential tool for early non-invasive diagnosis of CRC.
The Analyst 06/2011; 136(11):2252-9. · 4.23 Impact Factor
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ChemBioChem 03/2011; 12(6):845-9. · 3.94 Impact Factor
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ABSTRACT: In pyrosequencing chemistry, four cascade enzymatic reactions with the catalysis of polymerase, adenosine triphosphate (ATP) sulfurylase, luciferase, and apyrase are employed. The sensitivity of pyrosequencing mainly depends on the concentration of luciferase which catalyzes a photoemission reaction. However, the side-reaction of adenosine 5' phosphosulfate (APS, an analogue of ATP) with luciferase resulted in an unavoidable background signal; hence, the sensitivity cannot be much higher due to the simultaneous increase of the background signal when a larger amount of luciferase is used. In this study, we demonstrated a sensitive pyrosequencing using a large amount of ATP sulfurylase to lower the concentration of free APS in the pyrosequencing mixture. As the complex of ATP sulfurylase and APS does not react with luciferase, a large amount of luciferase can be used to achieve a sensitive pyrosequencing reaction. This sensitivity-improving pyrosequencing chemistry allows the use of an inexpensive light sensor photodiode array for constructing a portable pyrosequencer, a potential tool in a point-of-care test (POCT).
Analytical Chemistry 03/2011; 83(9):3600-5. · 5.86 Impact Factor
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Angewandte Chemie International Edition 11/2010; 50(32):7395-8. · 13.45 Impact Factor
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ABSTRACT: A general method is described to analyze pathogen DNAs in homogenous solution by a novel Bio-MassCode probe, using a small organic (namely disulfide) "reporter mass" that self-assembled in oligonucleotide-modified gold nanoparticles. With conventional MALDI TOF MS, the assay simultaneously detected DNAs for HIV, HBV, HCV, and TP with extraordinarily high sensitivity in the low attomolar (10(-18) M) range without any other amplification.
Chemical Communications 09/2010; 46(43):8288-90. · 6.17 Impact Factor
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ABSTRACT: 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.
Analytical and Bioanalytical Chemistry 07/2010; 397(5):1937-45. · 3.78 Impact Factor
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ABSTRACT: Most methods used for gene expression analysis are based on dye-labeling, which requires costly instruments. Recently a dye-free gene expression analysis method-SRPP (Sequence-tagged reverse-transcription polymerase chain reaction coupled with pyrosequencing) was developed to compare relative gene expression levels in different tissues, but the throughput of the SRPP assay is very limited due to the use of a photomultiplier tube (PMT)-based pyrosequencer for the detection. To increase the throughput of the SRPP assay, an inexpensive photodiode (PD) array-based bioluminescence analyzer (termed as "PD-based pyrosequencer") was coupled to SRPP; however the low sensitivity of PD limited the wide application of SRPP. To enable SRPP analyzing low abundance genes in clinical samples, sequence-tagged gene-specific primers instead of sequence-tagged poly (T)(n) primers were used for reverse-transcription, and the SRPP sensitivity was thus improved more than 10 times. This improvement compensates the sensitivity loss due to the use of PD in a pyrosequencer. The accurate determination of the expression levels of ten prognostic marker genes (AL080059, MMP9, EXT1, ORC6L, AF052162, C9orf30, FBXO31, IGFBP5, ESM1, and RUNDC1) differing between normal tissues and tumor tissues of breast cancer patients demonstrated that SRPP using gene-specific RT primers coupled with the PD array-based bioluminescence analyzer is reliable, inexpensive, and sensitive in gene expression analysis.
The Analyst 06/2010; 135(6):1315-9. · 4.23 Impact Factor
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ABSTRACT: 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, approximately 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 allele-specific 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.
Analytical Chemistry 03/2010; 82(5):2074-81. · 5.86 Impact Factor
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ABSTRACT: As conventional solid-phase amplification (SPA) on a two-dimensional slide has a low amplification capacity due to a limited amount of immobilized primers, we propose a three-dimensional SPA by immobilizing primers in hydrogel attached to a slide. One of the PCR primers, modified with an acrylamide group at the 5'-terminal, was copolymerized with both polyacrylamide gel and an acryl-modified glass slide, resulting in a high amplification capacity. The immobilization process was carried out by adding the catalysis reagent N,N,N',N'-tetramethylethylenediamine (TEMED) volatilized in vacuum, with uniform sample-concentration and gel-viscosity in the course of one-step nucleic acid immobilization. The porous structure of polyacrylamide gel, which allows PCR reagents such as Taq DNA polymerase, primers, dNTPs and DNA templates to freely enter the gel matrix, provides a homogeneous solution-mimicking environment for SPA on the interface or the inside of gel pads. Based on gel-based SPA, genotypes of different samples were accurately discriminated by either dual-color fluorescence hybridization or BAMPER (Bioluminometric Assay coupled with Modified Primer Extension Reactions). Pyrosequencing was also successfully carried out on SPA products. As the linkage between DNA molecules and gel is very strong, SPA products immobilized on gel pads could be reused several times if extended strands were removed by electrophoresis. Thus, the gel-based SPA provides a powerful tool for directly using on-chip amplicons for parallel detection.
The Analyst 12/2009; 134(12):2434-40. · 4.23 Impact Factor
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ABSTRACT: Recently, we have developed a method of adapter-ligation mediated allele-specific amplification (ALM-ASA) for simultaneously typing multiple single nucleotide polymorphisms (SNPs) at a low cost. We usually use agarose gel-electrophoresis for analyzing PCR products. As the processes of sampling and PCR can be carried out at a format of 96-well or 384-well, the throughput-bottleneck of whole process of ALM-ASA is only the agarose gel-electrophoresis. Here we improved the typing throughput of ALM-ASA by using a microplate array parallel gel electrophoresis (MAPGE) system, with which 96 amplicons can be detected at a time. By coupling with multiplexed preamplification, seven SNPs distributed on four different human genes (IL1A (549C>T), 1L1B (794C>T and 5277C>T), IL10 (2940G>A, 3203C>T, and 3430C>A), and TNFA (1431G>A)) were successfully typed. The optimization of allele-specific primers in ALM-ASA was performed by the software of "SNiPdesigner" which was designed especially for ALM-ASA. We also demonstrated that the specificity of ALM-ASA assay for SNP typing is superior to that of amplification refractory mutation system (ARMS).
Molecular Biotechnology 09/2009; 44(1):1-7. · 2.17 Impact Factor
