To achieve a microarray universal to any SNP, we proposed a new way to construct a genotyping–microarray by ligating a universal fluorescence-probe with SNP-encoded flaps cleaved from invasive reactions on a slide surface. Our proposed microarray is labor-saving and cost-saving in setting up a new experiment for genotyping multiple SNPs, which is related to personalized medicine.

To achieve a microarray universal to any SNP, we proposed a new way to construct a genotyping–microarray by ligating a universal fluorescence-probe with SNP-encoded flaps cleaved from invasive reactions on a slide surface. Our proposed microarray is labor-saving and cost-saving in setting up a new experiment for genotyping multiple SNPs, which is related to personalized medicine.

•Visualized genotyping of multiple targets was achieved by detecting multiplexed LAMP amplicons with series target-specific invasive reactions coupled with hybridization-induced assembly of gold nanoparticles.•The key steps of the method include sequence amplifier to amplify the multiple targets, allele identifier to recognize a targeted base in the multiplexed amplicons, and signal generator to yield red color from disconnected gold nanoparticles.•Since sequence amplifier and allele identifier occurs at a constant temperature, the method only needs a simple temperature controller, much suitable for source-limited regions to assist with gene-guided personalized medicine.Loop-mediated isothermal amplification (LAMP) is a well-developed DNA amplification method with an ultra-high sensitivity, but it is difficult to recognize a single-base difference (like genotyping) in target-specific amplicons by conventional detection ways, such as the intercalation of dyes into dsDNA amplicons or the increase of solution turbidity along with the polymerization process. To allow genotyping based on LAMP suitable for POCT (point-of-care testing) or on-site testing, here we proposed a highly specific and cost-effective method for detecting a single-base difference in LAMP amplicons. The method includes three key steps, sequence amplifier to amplify multiple fragments containing the single nucleotide polymorphisms (SNPs) of interest, allele identifier to recognize a targeted base in the amplicons by invasive reaction, and signal generator to yield signals by hybridization-induced assembly of oligonucleotide probe-modified gold nanoparticles. Because the allele identifier is sensitive to one base difference, it is possible to use multiplexed LAMP (mLAMP) to generate amplicon mixtures for multiple SNP typing. Genotyping of 3 different SNPs (CYP2C19*2, CYP2C19*3 and MDR1-C3435T) for guiding the dosage of clopidogrel is successfully carried out in a 3-plex LAMP on real clinical samples. As our method relies on the naked-eye detection and constant-temperature reaction, no expensive instrument is required for both target amplification and sequence identification, thus much suitable for inexpensive gene-guided personalized medicine in source-limited regions.

Non-invasive detection of trisomy 21 is a safe and effective way for prenatal diagnosis. Although using next generation sequencing technology can achieve non-invasive detection of chromosomal abnormalities, a more convenient and cost-effective method is preferable for routine clinical applications. Here, we proposed a novel method for the detection of trisomy 21 by accurately quantifying the slightly increased amount of chromosome 21 in cell-free DNA from maternal plasma using digital PCR. The segmental duplication fragments on chromosome 21 and chromosome 1 were employed as the detection target of digital PCR. As low as 10% cell-free fetal DNA of trisomy 21 fetus in maternal cell-free DNA was successfully detected. Three trisomy 21 samples were unambiguously picked up from 15 clinical samples, indicating that our method has the potential for non-invasive diagnosis of trisomy 21.

A new method for polymorphism detection of rs9263726 site on Psoriasis Susceptibility1 Candidate1 (PSORS1C1) was established based on the pyrosequencing technology and the improved method of genomic DNA extraction from whole blood. The detection limit of the method was 0.4 ng μL−1 genomic DNA. A total of 20 samples were detected by pyrosequencing, and the results were consistent with those of Sanger sequencing. By gene-sequencing of 683 clinical samples with pyrosequencing method, the polymorphism distribution of rs9263726 in Chinese was summarized as GG (87.6%), GA (11.7%), and AA (0.7%). Besides, 46 clinical samples were detected to analyze the relationship between rs9263726 genotype and HLA-B*5801 genotype, and the results showed that the sensitivity of the method was 100%, and the specificity was 91.3%. This study demonstrated a new method for HLA-B*58:01genotyping.This study demonstrated a new method for HLA-B*58:01 genotyping. Adopting pyrosequencing for detecting the genotypes of the locus rs9263726 rather than detecting HLA-B*58:01 through PCR-SBT allows to reduce the cost, shorten detection time, and avoid the occurrence of highly-fatal SJS/TEN syndrome induced by carrying HLA-B*58:01 genotype.

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 spectrometry-based metabolic profiling to predose plasma samples from 48 healthy volunteers. We determined the level of atorvastatin in plasma using liquid chromatography–tandem mass spectrometry. With the endogenous molecules, which showed a good correlation with pharmacokinetic parameters, a refined partial least-squares model was calculated based on predose 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 by showing better predictive capability for explaining individual differences than a 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.

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.

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.Invasive reaction-coupled nicking endonuclease assisted nanoparticles amplification (IR-NEANA) enables us to detect somatic mutations by naked eyes.

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.

•A portable bioluminescence analyzer for detecting the DNA sequence was developed.•The DNA sequences of eight genes of GMO were successfully detected.•The corresponding limit of detection (LOD) was 0.01% with 35 PCR cycles.•The results indicate the method is suitable for the field test of GMO analysis.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.

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.

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).

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.

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).Highlights► We proposed an IAC-based dipstick-type biosensor for quantifying pathogen DNA. ► Quantification was achieved by using an IAC with a sequence similar to the target. ► IAC can monitor the reliability of the whole process and avoid false negatives. ► Two-step PCR was used for quick and specific amplification.

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.

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.

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.

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.

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).

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.

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.

Streptococcus suis (S. suis for short) can cause a variety of infections in pigs, and the infections have brought about great losses in the swine industry and some cases of deaths in human beings. In order to rapidly diagnose and control the infections of S. suis, we designed a pyrosequencing-based assay to identify the serotypes of S. suis. In the assay, pyrosequencing is used to genotype most of the pathogenic serotypes of S. suis by detecting five informative regions on the Chaperonin 60 (cpn60) gene and one species-specific region on the 16S rRNA gene, and further a few undistinguished serotypes by pyrosequencing were finely discriminated by multiplex PCR of serotype-specific fragments on the cpsgene as well as species-specific fragments on the 16S rRNA gene. Through carefully designing the dispensing order of dNTP for each pyrosequencing reaction, the serotypes of S. suis could be discriminated by four pyrosequencing reactions within three hours. Five reference serotypes and three clinical strains were successfully detected and genotyped by our assay. The results indicated that our assay is a reliable, information-rich diagnostic method for the accurate detection ofS. suis serotypes.

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.

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 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.

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.

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).

In order to detect small amounts of mutants for early cancer diagnosis, we have developed the novel method of using amplicon-coated microbeads and single-molecule-PCR in water-in-oil emulsions, which we coupled with a new detection platform, the hydrogel bead-array, a 3-D polyacrylamidegel network used as a carrier to immobilize the beads.

Presently most techniques for gene expression analysis are based on a dye label. Here we describe a novel method for comparing gene expression levels among various tissues or cells by sequence-tagged reverse-transcription PCR coupled with pyrosequencing (termed “SRPP”). This method includes three steps: (i) reverse transcription of mRNA with sources-specific RT primers consisting of a tail at the 5′-end for supplying a common PCR priming site, a source-specific sequence in the middle, and a poly-T stretch plus several degenerate bases at the 3′-end for annealing the mRNA strand. (ii) PCR amplification of the templates produced by pooling sequence-labeled cDNAs equally from different sources. (iii) Decoding and quantification of the source-specific sequences tagged in the amplicons by pyrosequencing. The signal ratio in the pyrogram is proportional to the amounts of mRNAs among different sources. As the signal is detected by observing bioluminescence, neither dye, nor electrophoresis, or laser source was used. The expression levels of six kinds of genes (Cdk2ap2, Vps4b, Fas, Fos, Cdk4, and Actb) among the kidney, the brain, and the heart tissues of a mouse were accurately detected, suggesting that the new method is promising in quantitatively comparing gene expression levels among different sources at a low cost.

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.

It is an important way to understand the gene function by relatively comparing gene expression levels among different tissues or cells. For the moment, most of the methods for gene expression detection are based on dye labels. To establish a novel approach without using a dye label, a sequence-tagged reverse-transcription PCR coupled with pyrosequencing (SRPP) was proposed. In this technique, the gene from a source is labeled with a source-specific sequence by sequence-tagged reverse transcription (RT). Then PCR on the pools of each source-specific RT product was performed, and the source-specific amplicons were decoded by pyrosequencing. In the pyrogram, the sequence represents the gene source, and the peak intensity represents the relative expression level of the gene in the corresponding source. The accuracy of SRPP was confirmed by real-time quantitative PCR. Finally, the relative expression levels of the Egr1 gene among the diabetes model mice, obesity model mice, and normal mice were successfully detected. In comparison with real-time quantitative PCR, the advantages of SRPP include dye-free detection, inexpensive instruments, and simultaneous comparison of a given gene expressed in multiple sources.

To establish a simple method for preparing single-stranded DNA templates for pyrosequencing, the Linear-after-the-exponential (LATE)-PCR technology on the basis of Taq DNA polymerase without hot-start capacity was applied to amplify a 78-bp sequence (containing the SNP6 site), and the PCR-enhancing reagents (glycerol and BSA) were used to increase the efficiency and specialization, much more before the reagents A and B were designed to eliminate the impurity (limited primers, PPi, dNTPs and so on), and 1–2 μl LATE-PCR products with simply treatment can be used in pyrosequencing directly. Then, five SNPs related with human breast-cancers in the BRCA1 gene were investigated, and the programs had no nonspecific signals that were made of theoretic sequences. Moreover, the genotyping of the SNPs could also be distinguished easily. The results indicated that this method can be used to prepare high quality single-stranded DNA templates for pyrosequencing and allows pyrosequencing be lower in cost, simpler in operation, and easier in automation, and the cross-contamination from sample preparation was also reduced.

We described a novel approach to directly amplify genomic DNA from whole blood and dried blood spotted on filter paper without any DNA isolation by using the PCR buffer with a higher pH, which was optimized as pH 9.1–9.6. Direct PCR on blood treated with various anticoagulants showed that the buffer worked well with the blood treated by citrate, EDTA, or heparinate. DNA fragments with different lengths could be efficiently amplified directly from various forms of blood samples. By coupling the buffer with tetra-PCR, a “true” single-tube genotyping was realized by using whole blood or paper-dried blood as starting material.

•A digital quantification method based on hydrogel immobilized bead-array is proposed.•Gene methylation level in stool DNA is highly sensitive and accurate quantified.•The method shows a great potential for non-invasive screening of colorectal cancer.Aberrations of gene methylation in stool DNA (sDNA) is an effective biomarker for non-invasive colorectal cancer diagnosis. However, it is challenging to accurately quantitate the gene methylation levels in sDNA due to the low abundance and degradation of sDNA. In this study, a digital quantification strategy was proposed by combining emulsion PCR (emPCR) with hydrogel immobilized bead-array. The assay includes following steps: bisulfite conversion of sDNA, pre-amplification by PCR with specific primers containing 5′ universal sequences, emPCR of pre-amplicons with beaded primers to achieve single-molecular amplification and identification of hydrogel embedding beads coated with amplicons. The sensitivity and the specificity of the method are high enough to pick up 0.05% methylated targets from unmethylated DNA background. The successful detection of hypermethylated vimentin gene in clinical stool samples suggests that the proposed method should be a potential tool for non-invasive colorectal cancer screening.

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.

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.

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.

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.

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.