The effect of multiple primer–template mismatches on quantitative PCR accuracy and development of a multi-primer set assay for accurate quantification of pcrA gene sequence variants

Quantitative PCR (qPCR) is a critical tool for quantifying the abundance of specific organisms and the level or expression of target genes in medically and environmentally relevant systems. However, often the power of this tool has been limited because primer–template mismatches, due to sequence variations of targeted genes, can lead to inaccuracies in measured gene quantities, detection failures, and spurious conclusions. Currently available primer design guidelines for qPCR were developed for pure culture applications, and available primer design strategies for mixed cultures were developed for detection rather than accurate quantification. Furthermore, past studies examining the impact of mismatches have focused only on single mismatches while instances of multiple mismatches are common. There are currently no appropriate solutions to overcome the challenges posed by sequence variations. Here, we report results that provide a comprehensive, quantitative understanding of the impact of multiple primer–template mismatches on qPCR accuracy and demonstrate a multi-primer set approach to accurately quantify a model gene pcrA (encoding perchlorate reductase) that has substantial sequence variation. Results showed that for multiple mismatches (up to 3 mismatches) in primer regions where mismatches were previously considered tolerable (middle and 5′ end), quantification accuracies could be as low as ~ 0.1%. Furthermore, tests were run using a published pcrA primer set with mixtures of genomic DNA from strains known to harbor the target gene, and for some mixtures quantification accuracy was as low as ~ 0.8% or was non-detect. To overcome these limitations, a multiple primer set assay including minimal degeneracies was developed for pcrA genes. This assay resulted in nearly 100% accurate detection for all mixed microbial communities tested. The multi-primer set approach demonstrated herein can be broadly applied to other genes with known sequences.     

The ESR2 AluI 1730G>A (rs4986938) gene polymorphism is associated with fibrinogen plasma levels in postmenopausal women

Canavan disease (CD) is a neurodegenerative disorder usually presenting in the first six months of life. CD patients can be identified via elevated levels of N-acetyl-l-aspartate in the pattern of urinary organic acids assessed by gas chromatography-mass spectrometry. They are characterized by deficiency of aspartoacylase (aminoacylase 2; ASPA) due to mutations in the ASPA gene. Information on the molecular basis of CD is rather sparse. A lack of expression studies of ASPA mutant proteins in appropriate expression systems has prompted this investigation. Studies with overexpressed ASPA mutant proteins were carried out in the HEK293 cell line, which provides the authentic human machinery for posttranslational modifications. All ASPA mutants tested (ASPA Arg168His, ASPA Pro181Thr, ASPA Tyr288Cys, ASPA Phe295Ser, and ASPA Ala305Glu) showed loss of ASPA activity, which can be explained by the intramolecular effects of the mutations in the enzyme. The mutation p.Phe295Ser even leads to absent ASPA mRNA expression, as revealed by quantitative real-time PCR. Using this approach, ASPA gene expression analysis yielded high levels of human ASPA gene expression not only in brain and kidney, but also in lung and liver. More information of ASPA localization in human organs and detailed characterization of mutations leading to a deficiency of ASPA can contribute to a better understanding of this inborn error of metabolism.     

Linking microbial community structure to membrane biofouling associated with varying dissolved oxygen concentrations

In order to elucidate how dissolved oxygen (DO) concentration influenced the generation of extracellular polymeric substance (EPS) and soluble microbial products (SMP) in mixed liquor and biocake, 16S rDNA fingerprinting analyses were performed to investigate the variation of the microbial community in an aerobic membrane bioreactor (MBR). The function of microbial community structure was proved to be ultimately responsible for biofouling. Obvious microbial community succession from the subphylum of Betaproteobacteria to Deltaproteobacteria was observed in biocake. High concentration of EPS in biocake under the low DO concentration (0.5 mg L⁻¹) caused severe biofouling. The correlation coefficient of membrane fouling rate with EPS content in biocake (0.9941-0.9964) was much higher than that in mixed liquor (0.6689-0.8004).     

Chibby functions in Xenopus ciliary assembly,embryonic development,and the regulation of gene expression

Wnt signaling and ciliogenesis are core features of embryonic development in a range of metazoans. Chibby (Cby), a basal-body associated protein, regulates β-catenin-mediated Wnt signaling in the mouse but not Drosophila. Here we present an analysis of Cby?s embryonic expression and morphant phenotypes in Xenopus laevis. Cby RNA is supplied maternally, negatively regulated by Snail2 but not Twist1, preferentially expressed in the neuroectoderm, and regulates β-catenin-mediated gene expression. Reducing Cby levels reduced the density of multiciliated cells, the number of basal bodies per multiciliated cell, and the numbers of neural tube primary cilia; it also led to abnormal development of the neural crest, central nervous system, and pronephros, all defects that were rescued by a Cby-GFP chimera. Reduction of Cby led to an increase in Wnt8a and decreases in Gli2, Gli3, and Shh RNA levels. Many, but not all, morphant phenotypes were significantly reversed by the Wnt inhibitor SFRP2. These observations extend our understanding of Cby?s role in mediating the network of interactions between ciliogenesis, signaling systems and tissue patterning.     

Improving PCR and qPCR detection of hydrogenase A (<Emphasis Type="Italic">hyd</Emphasis>A) associated with Clostridia in pure cultures and environmental sludges using bovine serum albumin

Detection of hydA genes of Clostridia spp. using degenerative and species specific primers for C. butyricum were optimized by the addition of bovine serum albumin (BSA) to polymerase chain reaction (PCR) and quantitative PCR (qPCR) reactions. BSA concentrations ranging from 100 to 400 ng/μl were examined using pure cultures and a variety of environmental samples as test targets. A BSA concentration of 100 ng/μl, which is lower than previously reported in the literature, was found to be most effective in improving the detection limit. The brightness of amplicons with 100 ng/μl BSA increased in ethidium bromide-treated gels, the minimum detection limit with BSA was at least one log greater, and cycle threshold (C _T) values were lower than without BSA in qPCR indicating improved detection of target deoxyribonucleic acid for most samples tested. Although amplicon visualization was improved at BSA concentrations greater than or equal to 100 ng/μl, gene copy numbers detected by qPCR were less, C_T values were increased, and T _m values were altered. SYBR Green dissociation curves of qPCR products of DNA from pure culture or sludge samples showed that BSA at 100 ng/μl reduced the variability of peak areas and T _m values.     

中华蜜蜂哺育蜂与采集蜂行为转变相关基因的表达差异研究

【目的】文章旨在寻找中华蜜蜂Apis cerana cerana哺育蜂与采集蜂行为转变相关的重要基因。【方法】选取了8群中华蜜蜂,分别采集哺育蜂和采集蜂,然后通过实时荧光定量PCR对哺育蜂和采集蜂头部中mrjps、Ache、CSP3等22个基因的表达进行了分析。【结果】实验结果表明:mrjp3、mrjp7、Ache、LOC406142、Hbg3、Mblk-1、Ef-1a-f1、TpnCIIIa、Wat、Ant基因在哺育蜂和采集蜂中表达差异极显著(P<0.01),mrjp1、mrjp2、mrjp4、LOC406114、CSP3、Dop1、Jhe、Oa1、Per、TpnT基因在哺育蜂和采集蜂中表达差异显著(0.010.05)。【结论】mrjp3、mrjp7、Ache、LOC406142、Hbg3、Mblk-1、Ef-1a-f1、TpnCIIIa、Wat、Ant、mrjp1、mrjp2、mrjp4、LOC406114、CSP3、Dop1、Jhe、Oa1、Per、TpnT这些表达差异显著的基因很可能与中华蜜蜂哺育蜂与采集蜂的行为转变有关。本研究的结果为我们更好地认识理解蜜蜂行为转变的机制提供思路。     

Trichosanthin-induced specific changes of cytoskeleton configuration were associated with the decreased expression level of actin and tubulin genes in apoptotic Hela cells

Trichosanthin (TCS) possesses a broad spectrum of biological and pharmacological activities, including anti-cancer activities through apoptosis pathway. However, little is known about the effects of TCS on the cytoskeleton configuration and expression of actin and tubulin genes in Hela cell apoptosis. In the present study, apoptotic cytoskeleton structures were observed by confocal immunofluorescence microscopy, absolute amounts of actin and tubulin subunit mRNAs were determined by quantitative real-time PCR assays (QRT-PCR). Our results showed that the execution phase of cell apoptosis was a highly coordinated process of cellular reorganization, depolymerized microfilaments (MFs) accumulated in the coarsened cytoplasm and apoptotic bodies, followed by the formation of a ring microtubule (MT) structure beneath the plasma membrane. Importantly, apoptosis occurred by a suppression of actin and tubulin subunit gene expression. In particular, a rapid decrease in the amounts of gamma-actin mRNA preceded that of beta-actin; alpha- and beta-tubulin mRNAs were subsequently down-regulated in the later stage of Hela cell apoptosis. These results suggested that the execution of Hela cell apoptosis induced by TCS accompanied the specific changes of cytoskeleton configuration and, significantly, decreased the expression level of actin and tubulin subunit genes in different stages.     

Molecular characterization and expression analysis of ubiquitin-activating enzyme E1 gene in Citrus reticulata

Ubiquitin-activating enzyme E1 (UBE1) catalyzes the first step in the ubiquitination reaction, which targets a protein for degradation via a proteasome pathway. UBE1 plays an important role in metabolic processes. In this study, full-length cDNA and DNA sequences of UBE1 gene, designated CrUBE1, were obtained from ‘Wuzishatangju’ (self-incompatible, SI) and ‘Shatangju’ (self-compatible, SC) mandarins. 5 amino acids and 8 bases were different in cDNA and DNA sequences of CrUBE1 between ‘Wuzishatangju’ and ‘Shatangju’, respectively. Southern blot analysis showed that there existed only one copy of the CrUBE1 gene in genome of ‘Wuzishatangju’ and ‘Shatangju’. The temporal and spatial expression characteristics of the CrUBE1 gene were investigated using semi-quantitative RT-PCR (SqPCR) and quantitative real-time PCR (qPCR). The expression level of the CrUBE1 gene in anthers of ‘Shatangju’ was approximately 10-fold higher than in anthers of ‘Wuzishatangju’. The highest expression level of CrUBE1 was detected in pistils at 7 days after self-pollination of ‘Wuzishatangju’, which was approximately 5-fold higher than at 0 h. To obtain CrUBE1 protein, the full-length cDNA of CrUBE1 genes from ‘Wuzishatangju’ and ‘Shatangju’ were successfully expressed in Pichia pastoris. Pollen germination frequency of ‘Wuzishatangju’ was significantly inhibited with increasing of CrUBE1 protein concentrations from ‘Wuzishatangju’.