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To avoid sequencing error resulting from use of apyrase in conventional 4- enzyme pyrosequencing system, a non-apyrase 3-enzyme pyrosequencing system with a better performance of quantitative analysis was established. The method is to immobilize biotinylated DNA template, ATP sulfurylase and luciferase on streptavidin-coated magnetic beads for pyrosequencing. After pyrosequencing, ATP produced from the pyrosequencing reaction and excess dNTPs were removed by magnetic separation technique; another dNTP was then dispensed for sequencing reaction, and the components interfering with the next circle of pyrosequencing reaction were removed by the same way, achieving the circular sequencing. This new system can accurately measure base sequences of a target DNA template, and also can quantitatively determine the relative ratio of two alleles. The allele ratios in two SNPs (rs1042917 and rs4818219) having a higher heterozygote rate on chromosome 21 were successfully detected for 16 normal samples and 8 clinical samples from Down's syndrome patients. The results can accurately demonstrate whether or not the target sample has equal copies of chromosome 21 from mother and father. This paper established a non-apyrase 3-enzyme pyrosequencing method, which owns a good perform-ance of quantitative analysis. The method is especially suitable to allelic quantification of an SNP, enabling the rapid diagnosis of Down's syndrome by analyzing allele ratio of SNPs on chromosome 21.

A structure kinetic model was established for describing the relationship among cell growth, genistein formation and substrate utilization in suspension culture of Maackia amurensis. In this model, sucrose uptake, structure components production, intermediate matter changes, cell respiration loss and secondary metabolite (genistein) production were all predicted. The parameter sensitivity test was indicated that intermediate matter self-catalysis rate constant (k(b1)), structure component synthesis rate constant (k(b2)) and secondary metabolite synthesis rate constant (k(p)) were the most sensitive parameters for this model. If k(b1), k(b2) or k(p) are changed by 10%, the range of the aim function value would be changed by 12.8%, 4.61% and 2.54%, respectively. Adjustment of the other parameters only aroused a change of less than 0.5% in the aim function value. The predicted values were in good agreement with those obtained experimentally.

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.

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.

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.

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.

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.

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.

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.

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.

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.

To detect low levels of microorganism by bioluminescence assay, the reaction of ATP amplification catalyzed by ADK (adenylate kinase) combined with PPK (polyphosphate kinase) can be employed. However, the endogenous ADP bound to PPK is a background source and interfere the effective detection of low levels of exogenous ATP. We expressed a fusion protein of PPK and ADK and established a new method to decrease the background signal.The genes of PPK and ADK were amplified by PCR and cloned into vector pET28a (+) to provide a recombinant expression plasmid pET28a (+)-PPKADK to prepare the fusion protein. Apyrase was immobilized on the surface of magnetic beads coated with polyurethane to provide Beads-apyrase to eliminate background caused by ADP bound to PPK-ADK. The exogenous ATP and microorganism were also detected by using ATP amplification reaction coupled with bioluminescence assay.The purified fusion protein showed both ADK and PPK activities. Beads-apyrase could eliminate ADP contamination conveniently and effectively, thus less than 1 fmol of ATP was detected by ATP amplification reaction coupled with bioluminescence assay. Using ATP amplification reaction, the sensitivity of bioluminescence assay was 100-fold than that of normal bioluminescence assay without ATP amplification.Beads-apyrase is an effective tool to eliminate the background of the reaction of ATP amplification. The sensitivity of bioluminescence assay was increased significantly with concomitant use of ATP amplification and bioluminescence assay.

Pyruvate phosphate dikinase (PPDK; EC 2.7.9.1) is found in certain microorganisms and plants, and catalyzes the conversion of AMP, PPi and phosphoenolpyruvate (PEP) to ATP, Pi and pyruvate. Using the genomic DNA of Microbispora rosea subsp. aerata as the template, a DNA fragment encoding the gene PPDK was amplified by PCR and inserted into the expression vector pET28a(+), yielding pET28a (+)-PPDK. The E. coli BL21 (DE3) was transformed with the pET28a (+)-PPDK. After inducing with IPTG, the E. coli BL21 (DE3) [pET28a (+)-PPDK] expressed recombinant PPDK fused to an N-terminal sequence of 6-His Tag. The molecular weight of PPDK was estimated to be 101 kD by SDS-PAGE. The PPDK was purified by His * Bind Resin affinity chromatography and ultrafiltration using 10 kD cut-off membrane. The successful application of PPDK in pyrosequencing was also demonstrated.

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.

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.

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.

Engineered endonucleases are a powerful tool for editing DNA. However, sequence preferences may limit their application. We engineer a structure-guided endonuclease (SGN) composed of flap endonuclease-1 (FEN-1), which recognizes the 3′ flap structure, and the cleavage domain of Fok I (Fn1), which cleaves DNA strands. The SGN recognizes the target DNA on the basis of the 3′ flap structure formed between the target and the guide DNA (gDNA) and cut the target through its Fn1 dimerization. Our results show that the SGN, guided by a pair of gDNAs, cleaves transgenic reporter gene and endogenous genes in zebrafish embryonic genome. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-1038-5) contains supplementary material, which is available to authorized users.