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Conventional assays for identifying polymorphism of aldehyde dehydrogenase 2 (ALDH2) gene (rs671) are based on electrophoresis, fluorescence-labeled probes or sequencing, which are laborious and expensive. Herein, we proposed a visualized detection of ALDH2 genotype by PCR-based serial invasive reaction coupled with gold nanoparticle probe (GNP) assembling, in which target DNAs were firstly amplified by PCR, and then the alleles on the amplicons were identified by serial invasive reaction, and finally the genotyping results could be read out with naked eyes by observing the color changes caused by GNP assembling. If the allele-specific invasive probe matched the target sequence, the hairpin probe could be cleaved into two parts by Flap endonuclease 1 (FEN1). As a result, the GNPs could not be aggregated by the hairpin probe, keeping the solution red. Otherwise, the intact hairpin probe caused the aggregation of GNPs, leading to the color change from red to purple or clear. Therefore, the genotyping result could be read out with naked eyes by observing the color changes of the reaction. As low as 80 pg of genomic DNA could be accurately genotyped, and the genotyping results of 207 clinical samples were consistent with that of pyrosequencing, indicating that the proposed method has great potential in genotyping of personalized medicine-related single nucleotide polymorphism.

Chromosomal aberration mostly occurs in chromosomes 21, 18 and 13, with an incidence approximately 1 out of 160 live births in humans, therefore making prenatal diagnosis necessary in clinics. Current methods have drawbacks such as time consuming, high cost, complicated operations and low sensitivity. In this paper, a novel method for rapid and accurate prenatal diagnosis of aneuploidy is proposed based on pyrosequencing, which quantitatively detects the peak height ratio (PHR) of different bases of segmental duplication. A direct polymerase chain reaction (PCR) approach was undertaken, where a small volume of amniotic fluid was used as the starting material without DNA extraction. Single-stranded DNA was prepared from PCR products and subsequently analyzed using pyrosequencing. The PHR between target and reference chromosome of 2.2 for euploid and 3:2 for a trisomy fetus were used as reference. The reference intervals and z scores were calculated for discrimination of aneuploidy. A total of 132 samples were collected, within trisomy 21 (n = 11), trisomy 18 (n = 3), trisomy 13 (n = 2), and unaffected controls (n = 116). A set of six segmental duplications were chosen for analysis. This method had consistent results with karyotyping analysis, a correct diagnosis with 100% sensitivity and 99.9% specificity.

Understanding the community structure of soil microbes is required to evaluate the potential effects of genetically modified (GM) plants on ecological environments. Bacterial communities in soil planted with conventional cotton (CC) and transgenic cultivar (TC) in a natural ecosystem for three years were characterized by 454 pyrosequencing of the V3-V5 and V5-V9 regions of 16S rDNA from June to September 2013. V3-V5 and V5-V9 regions yielded a total of 12,848 and 10,541 OTUs, respectively. The V5-V9 amplicon was additionally used to detect phyla that were poorly sequenced by V3-V5 (such as Chlamydiae, Crenarchaeota and Archaea). Among the species detected by each primer pair, 46% of the species identified from V3-V5 and 60% of those identified from V5-V9 were detected by both primer pairs. Although distinct bacterial compositions existed between the two amplified regions, statistical analysis revealed no significant difference in the diversity indexes or phylogenetic patterns in TC versus compared to those in the CC control. Further, clustering analysis in both regions indicated that there was no unambiguous aggregation in TC compared to that in CC control. Of all 26 phyla detected by both regions, each region detected 2 distinct phyla exhibiting significant variations in abundance. The species unique to each treatment field accounted for less than 27% of all species and were rare taxa (abundance0.15%). However, a small fraction of diagnostic taxa with specific ecological functions differed significantly between TC and CC. These differences were not driven by any obvious environmental factors. The results established a comprehensive inventory of the bacterial communities associated with GM plants and indicated that transgenic cotton may not significantly affect soil microorganisms compared with conventional cotton over a three-year period. Furthermore, diagnostic taxa were provided for monitoring the perturbation in soil, but further verification in future studies is required.

A method was developed for genotyping of single nucleotide polymorphism (SNP) by real-time polymerase chain reaction (PCR) coupled with high specific invader assay. The concentrations of flap endonuclease 1 (FEN1 enzyme) and wild type detection probe were optimized to reduce the background signal. Under the optimal conditions including 1×buffer, 250 μM dNTPs, 0.5 μM each PCR primer, 0.05 μM invasive oligo probe, 0.125 μM wild detection probe, 0.5 μM mutant detection probe, 0.25 μM each fluorescence resonance energy transfer (FRET) probe, 1 U Taq DNA polymerase, 1.5 U FEN1, and approximate 40-200 ng of genomic DNA, the background signals of wild-type sample and mutant-type sample were dramatically decreased and the background interference to the esults was thus eliminated. The reaction program was initial denaturation at 95 °C for 3 min, followed by 10 cycles (95 °C for 20 s, 67 °C for 60 s, 70 °C for 30 s) and 35 cycles (95 °C for 20 s, 63 °C for 60 s, read the fluorescence signal, 70 °C for 30 s). In the experiment, a total of 21 cases of ALDH2*2, 19 cases of CYP2C19*2 and 19 cases of CYP2C19*3 were analyzed by the established method, and the results showed that the genotypes of ALDH2*2 were 10 cases of GG homozygote, 8 cases of GA heterozygote and 3 cases of AA homozygote; the genotypes of CYP2C19*2 were 9 cases of GG homozygote, 8 cases of GA heterozygote and 2 cases of AA homozygote; and the genotypes of CYP2C19*3 were 18 cases of GG homozygote and 1 case of GA heterozygote. These results were consistent with the detection results by pyrosequencing. The established method was specific, simple, short time-consuming and low cost, and could be used for SNP genotyping with no-pollution in single tube.

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.

Long-term growth of genetically modified plants (GMPs) has raised concerns regarding their ecological effects. Here, FLX-pyrosequencing of region I (18S) and region II (ITS1, 5.8S, and ITS2) rDNA was used to characterize fungal communities in soil samples after 10-year monoculture of one representative transgenic cotton line (TC-10) and 15-year plantation of various transgenic cotton cultivars (TC-15mix) over four seasons. Soil fungal communities in the rhizosphere of non-transgenic control (CC) were also compared. No notable differences were observed in soil fertility variables among CC, TC-10, and TC-15mix. Within seasons, the different estimations were statistically indistinguishable. There were 411 and 2 067 fungal operational taxonomic units in the two regions, respectively. More than 75% of fungal taxa were stable in both CC and TC except for individual taxa with significantly different abundance between TC and CC. Statistical analysis revealed no significant differences between CC and TC-10, while discrimination of separating TC-15mix from CC and TC-10 with 37.86% explained variance in PCoA and a significant difference of Shannon indexes between TC-10 and TC-15mix were observed in region II. As TC-15mix planted with a mixture of transgenic cottons (Zhongmian-29, 30, and 33B) for over 5 years, different genetic modifications may introduce variations in fungal diversity. Further clarification is necessary by detecting the fungal dynamic changes in sites planted in monoculture of various transgenic cottons. Overall, we conclude that monoculture of one representative transgenic cotton cultivar may have no effect on fungal diversity compared with conventional cotton. Furthermore, the choice of amplified region and methodology has potential to affect the outcome of the comparison between GM-crop and its parental line.

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.

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.

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.

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.

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.