Multiple displacement amplification (MDA), a most popular isothermal whole genome amplification (WGA) method, suffers the major hurdle of highly uneven amplification, thus, leading to many problems in approaching biological applications related to copy-number assessment. In addition to the optimization of reagents and conditions, complete physical separation of the entire reaction system into numerous tiny chambers or droplets using microfluidic devices, has been proven efficient to mitigate this amplifying bias in recent works. Here, we present another MDA advance, microchannel MDA (μcMDA), which decentralizes MDA reagents throughout a one-dimensional slender tube. Due to the double effect from soft partition of high molecular-weight DNA molecules and less-limited diffusion of small particles, μcMDA is shown to be significantly effective at improving the amplification uniformity, which enables us to accurately detect single nucleotide variants (SNVs) with higher efficiency and sensitivity. More importantly, this straightforward method requires neither customized instruments nor complicated operations, making it a ready-to-use technique in almost all biological laboratories.

Understanding of the bacterial community structure in drinking water resources helps to enhance the security of municipal water supplies. In this study, bacterial communities were surveyed in water and sediment during a heavy cyanobacterial bloom in a drinking water resource of Lake Taihu, China. A total of 325,317 high-quality sequences were obtained from different 16S ribosomal RNA (rRNA) regions (V3, V4, and V6) using the Miseq sequencing platform. A notable difference was shown between the water and sediment samples, as predominated by Cyanobacteria, Proteobacteria, and Actinobacteria in the water and Proteobacteria, Chloroflexi, and Verrucomicrobia in the sediment, respectively. The LD12 family dominated the water surface and was tightly associated with related indicators of cyanobacterial propagation, indicating involvement in the massive proliferation of cyanobacterial blooms. Alternatively, the genus Nitrospira dominated the sediment samples, which indicates that nitrite oxidation was very active in the sediment. Although pathogenic bacteria were not detected in a large amount, some genera such as Mycobacterium, Acinetobacter, and Legionella were still identified but in very low abundance. In addition, the effects of different V regions on bacterial diversity survey were evaluated. Overall, V4 and V3 were proven to be more promising V regions for bacterial diversity survey in water and sediment samples during heavy water blooms in Lake Taihu, respectively. As longer, cheaper, and faster DNA sequencing technologies become more accessible, we expect that bacterial community structures based on 16S rRNA amplicons as an indicator could be used alongside with physical and chemical indicators, to conduct comprehensive assessments for drinking water resource management.

Designed printed circuit boards (PCBs) are alternative substrates for master mold construction of microfluidic devices. However, the rough supportive material molds rough replicas and causes difficulty in device sealing. To overcome this difficulty, a copper layer is used to generate a smooth surface. Some other researchers have used thermoplastic elastomers, which are typically much easier to bond, instead of the general polymer polydimethysiloxane (PDMS). This study presents an extremely simple approach for fabricating PDMS-based microfluidic devices using PCBs as masters. Unlike those reported in the previous studies, commoditized PCBs fabricated at a common PCB manufactory were directly used as the master molds. Two layers of semi-cured silicone of a distinct base to curing agent ratios were bonded together by additional curing. Efficient bonding was accomplished and avoided insufficient adhesion due to the rough surfaces of the PDMS replicas. Highly monodisperse droplets with polydispersity values smaller than 1% were stably formed using the easy fabricated devices. Essential operations in droplet microfluidics were reliably conducted in the PDMS-based devices. Moreover, the droplets were orderly sorted by the microstructures in the fabricated multi-height devices. The fabrication process provided a simple, convenient and reliable approach to prepare the general polymer PDMS-based microfluidic devices with a minimal requirement for equipment.

•A breathing-based isothermal emulsion amplification (BIEA) method was developed.•BIEA showed excellent properties compared with conventional amplification method.•Terminal breathing of DNA duplex was firstly used in emulsion amplification.The reaction temperature is one of the main factors that affect the stability of emulsion PCR (emPCR). Focusing on this point, we applied the “DNA breathing” mechanism in BEAMing (Bead, Emulsion, Amplification, and Magnetic) and proposed a more stable emulsion amplification method. Compared to the conventional emPCR, this method provided excellent results. Firstly, more stable emulsion system resulted in higher percentage of single-molecular amplifications (73.17%). Secondly, an ordinary temperature-controlling device was enough. Our outcome showed that the reaction temperature of this method was not strict so that the ordinary temperature-controlling device was enough for it (the heat block sets vs. the PCR instrument: 13.140 ± 0.110 vs. 13.008 ± 0.039, P = 0.120). Thirdly, the single-biotinylated emP1 coated streptavidin beads were stable enough to be used for this method (the control temperature vs. the reaction temperature: 2967.91 ± 409.045 vs. 3026.22 ± 442.129, P = 0.334), which could replace the double-biotinylated emP1 coated beads and was benefit for saving cost. In conclusion, the method presented here with stable emulsion system, simplified temperature-controlling device, and decreased investment would be a highly streamlined and inexpensive option for future single-molecular amplification based researches.Download high-res image (202KB)Download full-size image

Compared with microfluidic devices, the fabrication of structure-controllable and designable nanochannel devices has been considered to have high costs and complex procedures, which require expensive equipment and high-quality raw materials. Exploring fast, simple and inexpensive approaches in nanochannel fabrication will be greatly helpful to speed up laboratory studies of nanofluidics. Here we developed a simple and inexpensive approach to fabricate a nanochannel device with a glass/epoxy resin/glass structure. The grooves were engraved using a UV laser on an aluminum sacrificial layer on the substrate glass, and epoxy resin was coated on the substrate and stuffed fully into the grooves. Another glass plate with holes for fluidic inlets and outlets was bonded on the top of the resin layer. The nanochannels were formed by etching thin sacrificial layers electrochemically. Meanwhile, the microstructures of the fluidic outlets and inlets could be fabricated simultaneously to the nanochannel formation. The total processing time for the simple nanochannel device took less than 10 hours. Optically transparent nanochannels with a depth of up to 20 nm were achieved. Nanofluidic behaviors in the nanochannels were observed under both optical and fluorescence microscopes.

Although the diploid nature has been observed for over 50 years, phasing the diploid is still a laborious task. The speed and throughput of next generation sequencing have largely increased in the past decades. However, the short read-length remains one of the biggest challenges of haplotype analysis. For instance, reads as short as 150 bp span no more than one variant in most cases. Numerous experimental technologies have been developed to overcome this challenge. Distance, complexity and accuracy of the linkages obtained are the main factors to evaluate the efficiency of whole genome haplotyping methods. Here, we review these experimental technologies, evaluating their efficiency in linkages obtaining and system complexity. The technologies are organized into four categories based on its strategy: (i) chromosomes separation, (ii) dilution pools, (iii) crosslinking and proximity ligation, (ix) long-read technologies. Within each category, several subsections are listed to classify each technology. Innovative experimental strategies are expected to have high-quality performance, low cost and be labor-saving, which will be largely desired in the future.

•The amplification-based technology with significant advantage in higher sensitivity is a key component of miRNA studies.•Advanced amplification-based methods cannot replace traditional ones to be conventionally used due to the lack of enough verification.•Several sample-specific normalizers have been validated, suggesting that a single universal normalizer for all sample types is unlikely to exist, yet the use of different combinations of several normalizers for different sample types may be more appropriate.Over the last two decades, the study of miRNAs has attracted tremendous attention since they regulate gene expression post-transcriptionally and have been demonstrated to be dysregulated in many diseases. Detection methods with higher sensitivity, specificity and selectivity between precursors and mature microRNAs are urgently needed and widely studied. This review gave an overview of the amplification-based technologies including traditional methods, current modified methods and the cross-platforms of them combined with other techniques. Many progresses were found in the modified amplification-based microRNA detection methods, while traditional platforms could not be replaced until now. Several sample-specific normalizers had been validated, suggesting that the different normalizers should be established for different sample types and the combination of several normalizers might be more appropriate than a single universal normalizer. This systematic overview would be useful to provide comprehensive information for subsequent related studies and could reduce the un-necessary repetition in the future.

•Templates are determined without directly measuring the base sequence in this method.•Templates are sequenced with the incorporation of AG/CT, AC/GT or AT/CG.•Templates will be sequentially decoded by two sets of encodings.•This method applies fewer cycles to obtain longer read length.•This method is able to be applied to differentiate nucleic acid sequences.We propose a real-time decoding sequencing strategy in which a template is determined without directly measuring base sequence but by decoding two sets of encodings obtained from two parallel sequencing runs. This strategy relies on adding a mixture of different two-base pair, A + G, C + T, A + C, G + T, A + T or C + G (abbreviated as AG, CT, AC, GT, AT, or CG), into the reaction each time. When a template is cyclically interrogated twice with any two kinds of dual mononucleotide addition (AG/CT, AC/GT, and AT/CG), two sets of encodings are obtained sequentially. The two sets of encodings allow for the bases to be sequentially decoded, moving from first to last, in a deterministic manner. This strategy applies fewer cycles to obtain longer read length compared to the traditional real-time sequencing strategy [1]. Partial rnpB gene was applied to verify the applicability of the decoding strategy via pyrosequencing. The results indicated that the sequence could be reconstructed by decoding two sets of encodings. Moreover, streptococcal strains could be differentiated by comparing signal intensity in each cycle and encoding size of each template. This strategy is likely to be applied to differentiate nucleic acid sequence as encoding size and signal intensity in each cycle vary with the base size and composition. Furthermore, it has the potential in building a promising strategy that could be utilized as an alternative to conventional sequencing systems.

Sequencing-by-ligation (SBL) is one of the next-generation sequencing methods for massive parallel sequencing. The ligated probes used in SBL should be accurately cleaved for a better ligation in the next cycle. Here, a novel kind of oligonucleotide probe that could be accurately cleaved at the given position was proposed. Deoxynucleoside phosphorothioates were introduced into the deoxyoxanosine-containing oligonucleotide probes in order to increase the cleavage accuracy of endonuclease V on double-stranded DNA templates. The results illustrated that incorporating deoxynucleoside phosphorothioates could greatly reduce the effect of the nonsynchronous sequencing primer, and the queried bases of the DNA templates were unambiguously identified with 5 cycles of sequencing ligations. Additionally, the read length can reach up to 25 bp with high accuracy. The SBL-based method is inexpensive, has high-throughput, and is easy to operate allowing massive scale-up, miniaturization and automation.

In this study, we present a global relative expression analysis of miRNAs in human miRNA gene cluster and family based on different selection schemes of isomiRs from deep sequencing data. Some divergence was observed between expression distributions based on sequence count of the most abundant isomiR and sum of all isomiR sequence counts, respectively. But the divergence showed less effect on expression patterns in miRNA gene cluster and family. Because of high sensitivity of sequencing technology and ambiguous distribution of common isomiRs among multicopy miRNA precursors and homologous genes, most gene clusters and families showed inconsistent expression levels. Inconsistent expression pattern implied expression diversification in vivo although they maybe showed close relationship during transcription and/or gene regulation. Some gene clusters and families generated mature miRNAs from the same arms and even with the same lengths, especially in gene family. The interesting expression and length distribution maybe implied strict regulation during pre-miRNA processing through cleavage of Dicer and Drosha. The study showed that the most abundant isomiR may be a better marker, and global expression analysis of miRNA gene cluster and family provide clues for analyzing relative expression levels and understanding the function relationship and mechanism for expression regulation of miRNAs in regulation network.

Inherent problems exist with sequencing-by-synthesis (SBS) methods which use fluorescein-labeled nucleotide incorporation into a target template based on a polymerase chain reaction (PCR). These problems include lowering the cost of sequencing and the removal of fluorescence in DNA sequencing for further reading. How can these sequencing problems be resolved? We present a sequencing strategy which we call an extension-quenching-extension sequencing on a microarray based on a two-primer hyperbranched rolling circle amplification (HRCA). The microarray is a high throughput and low-cost tool.The template on a microarray for SBS was prepared by HRCA. The Cy 5-labeled deoxyribonucleoside triphosphate (dNTP) species were incorporated in the extension reactions. We discovered that copper (CuSO4) can quench the fluorescence in DNA sequencing because it exhibits an energy-transfer mechanism of quenching from the fluorescein to the bound Cu2+ ion. The fluorescein label needs to be destroyed after the readout by CuSO4 before further reading is possible.This paper describes the process used which discovered a successful combination of temperature, concentration, and duration of use for CuSO4 which successfully quenched the fluorescence. In this experiment, we used a known sequence as a template in order to provide a strategy for sequencing.

Massively parallel genomic DNA fragments display on chip plays a key role in the new generation DNA sequencing. Here, we developed a new technology to display the parallel genomic DNA fragment massively based on two-step reaction with Ф29 DNA polymerase. The genomic DNA fragments were firstly amplified by rolling-circle amplification (RCA) reaction in liquid phase, and then amplified further on the chip by the strand displacement of Ф29 DNA polymerase. In our experiments, through DNA colonies produced by two-step amplification reaction T7 genomic DNA fragments are displayed massively and parallely on the chip, which has been verified through hybridizing the probe labeled with fluorescence or extension reaction with fluorescent-dNTP. The significant difference of fluourescence signals between background and displayed DNA fragments could be obtained. Our results show that the method has good reproducibility in experiments, which may be hopeful to serve the high-throughput sequencing.

The combination of chromatin immunoprecipitation with sequencing (ChIP-Seq) is an effective method for obtaining an in vivo genome-wide profile of the interaction of a protein with DNA. With the dramatic development of high-throughput short sequencing technologies, several new algorithms have been developed to process ChIP-Seq. However, the reported analytical tools for ChIP-Seq based on size selection of immunoprecipitated (IPed) DNA fragments are mainly adopted on the Solexa system. As a sequencer with the highest throughput, few studies of ChIP-Seq based on SOLiD system have been reported. The main difference of the SOLiD and Solexa systems exists in the length of DNA fragments during preparing sequencing libraries. The SOLiD system has relatively short DNA fragments if it processes a further sonication of IPed DNA fragments in order to meet the length requirement of DNA fragments for emulsion-PCR (ePCR). This work aims to investigate the influences of DNA fragment length on data analysis from ChIP-Seq. Previous studies show that typical bimodal peaks can be observed in Solexa ChIP-Seq data, but based on the analysis of the real SOLiD ChIP-Seq data in this study, we found that there were no double peaks with apparent reads shift in a local enriched region and the local reads distribution of peaks were tested by normal distribution. Using real and simulated ChIP-Seq data, three main ChIP-Seq algorithms (CisGenome, SISSRs and MACS) have been investigated. We found that algorithms developed for processing ChIP-Seq data generated from Solexa library protocol, cannot efficiently capture the feature of the ChIP-Seq data from SOLiD library. Misuse of those analytical tools would be a possible reason for failure of ChIP-Seq on the SOLiD system. Therefore, a new ChIP-Seq analytical strategy for an extra-sonication of IPed DNA fragments needs to be developed.

The microfossils from Neoproterizoic Doushantuo Formation, Southeast China, include multicellular animal and algae fossils with exquisite cellular or even subcellular structure preserved, which show the beginning of biodiversity in the Precambrian ocean about 600 million years ago. The fossil cells can fluoresce when excited by laser light and have a steady fluorescence emission in the visible spectrum. The organic matter was thought as the fluorescence source. However, our observation of the microfossils from Doushantuo Formation, Weng‘an reveals another fluorescence source. We found by scanning electron microscopy and transmission light microscopy that the fossil cells with fluorescence are composed of small crystals in a naturally self-assembled manner. Through energy dispersive spectroscopy we know that the crystal is mainly composed of Ca, P, O, and F elements. Furthermore, we performed powder X-ray diffraction to confirm that the crystal compound is Ca5(PO4)3F. Therefore, we consider that crystallization of Ca5(PO4)3F is the main reason that causes the fossil cells to illuminate.

A method for determining methylation density of target CpG islands has been established. In the method, DNA microarray was prepared by spotting a set of PCR products amplified from bisulfite-converted sample DNAs. The PCR products on the microarray were treated by SssI methyltransferase and labeled with TAMRA fluorescence. A recombinant, antibody-like methyl-CpG-binding protein labeled with Cy5 fluorescence was used to identify symmetrical methyl-CpG dinucleotide of the PCR products on the microarray. By use of a standard curve with control mixtures, the ratio of two fluorescence signals can be converted into percentage values to assess methylation density of targeted fragments. We obtained the methylation density of six CpG islands on the two tumor suppressor genes of CDK2A and CDK2B from seven cancer cell line samples and two normal blood samples. The validity of this method was tested by bisulfite sequencing. This method not only allows the quantitative analysis of regional methylation density of a set of given genes but also could provide information of methylation density for a large amount of clinical samples.

A sensitive approach for the qualitative detection of DNA-binding protein on the microarray was developed. DNA complexes in which a partial duplex region is formed from a biotin-primer and a circle single strand DNA (ssDNA) were spotted on a microarray. The endonuclease recognition site (ERS) and the DNA-binding sites (DBS) were arranged side by side within the duplex region. The working principle of the detection system is described as follows: when the DNA-binding protein capture the DBS, the endonuclease could not attach to the ERS, and the immobilized primer in the DNA complex could be extended along the circle ssDNA by rolling circle amplification (RCA). When no protein protects the DBS, the ERS could be attacked by the endonuclease and subsequently no rolling circle amplification occurs. Thereby we can detect the sequence specific DNA-binding activity with high-sensitivity due to the signal amplification of RCA.

The effects of various environmental factors in unfolding/refolding pathway of proteins on ANS fluorescence were investigated. In phosphate buffer of pH 7.4, ANS bound cetyl trimethyl ammonium bromide (CTMAB) and resulted in significant increase of ANS fluorescence and a sudden blue-shift in emission maximum wavelength. Sodium dodecyl sulfate (SDS) caused less increase in ANS fluorescence and less blue-shift in emission maximum wavelength than CTMAB and Tween 20. Tween 20, urea, polyethylene glycol 8000 (PEG 8000) and glycerol which contains hydrophobic groups also enhanced ANS fluorescence and caused their emission maximum wavelength blue-shift. For guanidine hydrochloride (GdmCl), due to its shorter hydrophobic chain, it caused less increase in ANS fluorescence and less decrease in emission maximum wavelength than CTMAB although GdmCl also has a positive charge. The results demonstrated the need for caution in interpreting enhancement of ANS fluorescence as an evidence for the existence of partially folded protein intermediates.

The interaction of 6-thioguanine (6-TG) and bovine serum albumin (BSA) in the absence and presence of denaturant (urea and Guanidine hydrochloride) was investigated by fluorescence spectroscopic techniques. Changes of fluorescence intensity both in F304 and in F348 of BSA reflected increasing participation of tyrosine fluorescence in the total emission with increasing denaturant, which indicated that energy transfer from tyrosyl residues to tryptophanyl residues became less efficient in the denatured tertiary protein structure. The quenching effects of 6-TG were shown not only on the native but also on the unfolded form of BSA. The quenching constants and binding constants were calculated from the fluorescence spectra of the BSA/6-TG complex both in the absence and presence of the denaturant. The data suggested that the quenching constants and binding constants of 6-TG for BSA decreased with increasing concentration of denaturant. The spectroscopic analysis also showed antidenaturant properties of 6-TG under both denaturant conditions.

Photonic crystal sensing materials have been validated that they are very sensitive to refractive index changes. Herein, three-dimensionally ordered macroporous (3DOM) (>50 nm) TiO2 inverse opal film has been fabricated by the self-assembly technique. Based on the TiO2 inverse opal film, the optical spectrometer was established for label-free immunosensor. The sensing performance of the 3DOM TiO2 was investigated using human IgG/goat anti-human IgG couple, which showed that the sensitivity of 3DOM TiO2 inverse opal film could reach to 1 μg mL−1 (equivalent to 1.5 pg mm−2) of protein concentration detection limit. The 3DOM TiO2 inverse opal has a large internal surface area, low fluorescence background and unique optical properties. These characteristics indicated the feasibility of 3DOM TiO2 inverse opal in label-free immunoassay.

Aberrant DNA methylation of CpG site in the gene promoter region has been confirmed to be closely associated with carcinogenesis. In the present study, a microarray-based methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) for parallel detecting changes of DNA methylation in cancer was developed. After modification by sodium sulfite, the unmethylated cytosine in the genomic DNA is converted to uracil while leaving the 5-methylcytosine unchanged, which can be detected by bifunctional primer carrying a unique sequence tag in addition to a locus-specific sequence. Because each locus has a distinct tag, the detecting reactions can be performed in a highly multiplexed fashion and the resulting product then be hybridized to the reverse complements of the sequence tags arrayed on a glass slide for methylation analysis. The calibration curves with the correlation coefficient >0.97 were established, which suggested that the method could be used in near-quantitative DNA methylation analysis. Two breast tumor-related genes (E-cad and p16) are successfully analyzed by two group primers (22 primers total), and the results are compatible with that of methylation-specific PCR (MSP). Our research proved that the method is simple and inexpensive, and could be applied as a high-throughput tool to quantitatively determine methylation status of the investigated genes.

We developed a protein microarray methodology that has the ability of serodiagnosis of IgM antibodies directed against TORCH pathogens. Six chemical surface modifications were validated by a dimension atomic force microscope (AFM) and contact angle measurement, agarose modified surface of which offered an appropriate platform for detecting IgM antibody. Further, signal amplification sensitivities on agarose modified microarrays were detected by Cy3-labeled biotin–streptavidin and immunogold-based assays. The detection limits of IgM antibody on the microarrays were 0.48 and 0.24 μg/ml, quantitatively equal to 0.25 and 12.5 pg, respectively, on each spot as ascertained by the two assays. Satisfactory linear correlations between the signal intensity and the logarithm of the IgM concentration were obtained. Finally, 60 serum samples characterized by a commercial ELISA were evaluated by the protein microarray. There were good concordances between the results of the protein microarray and ELISA assay for sorting of the TORCH infected sera (95.0% by fluorescence-based assay and 96.7% by immunogold-based assay). Clearly, the potential application of this protein microarray format facilitates clinical detection of not only the antibodies directed against TORCH pathogens but also other autoimmune diseases.

Aberrant DNA methylation of CpG site in the gene promoter region has been confirmed to be closely associated with carcinogenesis. In this present study, a new method based on the allele-specific extension on microarray technique for detecting changes of DNA methylation in cancer was developed. The target gene regions were amplified from the bisulfite treated genomic DNA (gDNA) with modified primers and treated with exonuclease to generate single-strand targets. Allele-specific extension of the immobilized primers took place along a stretch of target sequence with the presence of DNA polymerase and Cy5-labeled dGTP. To control the false positive signals, the hybridization condition, DNA polymerase, extension time and primers design were optimized. Two breast tumor-related genes (P16 and E-cadherin) were analyzed with this present method successfully and all the results were compatible with that of traditional methylation-specific PCR. The experiments results demonstrated that this DNA microarray-based method could be applied as a high throughput tool for methylation status analysis of the cancer-related genes, which could be widely used in cancer diagnosis or the detection of recurrence.

Polymethyl methacrylate (PMMA) spheres synthesized at boiling temperature were used to fabricate non-close-packed arrays by vertical lifting method. Scanning electron microscope (SEM) images demonstrated that these spheres could stack with square lattices parallel to the substrate and the neighboring spheres in the same layer were not contacted each other. In addition, the distance between the spheres can be controlled by thermal treatment. The method gave a new approach for extending the possibility of nanospheres lithograph and the creation of photonic band gap materials.

Aberrant DNA methylation of CpG islands is among the earliest and most frequent alterations in cancer. It is of great importance to develop simple and high-throughput methods of methylation analysis for earlier cancer diagnosis or the detection of recurrence. In this study, bisulfite-modified target DNA arrays were prepared on positively charged nylon membrane with two different procedures: fixing PCR products and fixing genomic DNA. First, a bisulfite PCR product array was prepared through fixing PCR products amplified in bisulfite sequencing primers from the bisulfite-modified genomic DNA of different clinical samples on membrane. Furthermore, bisulfite-modified genomic DNA of the different samples was directly fixed on membrane to fabricate bisulfite genomic DNA arrays. The two kinds of arrays were hybridized by probes labeled with digoxigenin, and the hybridization signals were obtained through chemiluminescent detection. The methylation statuses of the IGFBP7 gene for breast tumor and normal tissue samples and for normal human blood cell samples were detected successfully by the two procedures. It was shown that the methods are reliable and sensitive and that they have high potential in screening molecular methylation markers from a large number of clinical samples.

We report a method to perform electroless copper plating on glass that could generate a solid copper film. In this method, γ-mercaptopropyltrimethoxysilane (MPTS) was used to form self-assembled molecular layers on commercially available glass slides, which were subsequently activated by dipping them directly into colloidal Ag solution instead of conventional two-step method, SnCl2 sensitization followed by PdCl2 activation, and applied successfully for electroless Cu plating. Experimental characterizations showed that a thin layer of Ag colloids was anchored onto the glass surface through SAg bonds, resulting a quicker deposition of copper metal and a stronger adherence of copper film on MPTS-modified glass surface than those on conventional two-step modified glass.

The degenerate primer-based sequencing was developed by a synthesis method (DP-SBS) for high-throughput DNA sequencing, in which a set of degenerate primers are hybridized on the arrayed DNA templates and extended by DNA polymerase on microarrays. In this method, a different set of degenerate primers containing a given number (n) of degenerate nucleotides at the 3′-ends were annealed to the sequenced templates that were immobilized on the solid surface. The nucleotides (n+1) on the template sequences were determined by detecting the incorporation of fluorescent labeled nucleotides. The fluorescent labeled nucleotide was incorporated into the primer in a base-specific manner after the enzymatic primer extension reactions and nine-base length were read out accurately. The main advantage of the DP-SBS is that the method only uses very conventional biochemical reagents and avoids the complicated special chemical reagents for removing the labeled nucleotides and reactivating the primer for further extension. From the present study, it is found that the DP-SBS method is reliable, simple, and cost-effective for laboratory-sequencing a large amount of short DNA fragments.

High-throughput sequencing is a powerful tool for discovering and profiling microRNAs (miRNAs) to gain further insights into their biogenesis and function. Due to shorter size, short RNAs from deep sequencing dataset are prone to map to multiple loci with an equal number of mismatches, especially among multicopy miRNA precursors and homologous miRNA genes. Systematic analysis of SOLiD sequencing dataset showed that 37.94% short RNAs could simultaneously map to more than one miRNA precursor, and more short RNAs were found to have multiple genomic loci. Improper selection from candidate loci might lose some mapping information, influence miRNA expression profile or even mislead to identify novel miRNAs. A comprehensive study indicated several potential features for correction strategy: location and distribution of mismatches, quality values, expression profiles of multiple isomiRs (miRNA variants), miRNA* and moRs (miRNA-offset-RNAs) at candidate locus and in its flank sequence. Further studies should develop an approach to correct the widespread phenomenon of multiple mapping based on these features, and improve accuracy of profiling and discovering miRNAs.

In this study, a comparative analysis of the codon usage bias was performed in Aeropyrum pernix K1 and two other phylogenetically related Crenarchaeota microorganisms (i.e., Pyrobaculum aerophilum str. IM2 and Sulfolobus acidocaldarius DSM 639). The results indicated that the synonymous codon usage in A. pernix K1 was less biased, which was highly correlated with the GC3S value. The codon usage patterns were phylogenetically conserved among these Crenarchaeota microorganisms. Comparatively, it is the species function rather than the gene function that determines their gene codon usage patterns. A. pernix K1, P. aerophilum str. IM2, and S. acidocaldarius DSM 639 live in differently extreme conditions. It is presumed that the living environment played an important role in determining the codon usage pattern of these microorganisms. Besides, there was no strain-specific codon usage among these microorganisms. The extent of codon bias in A. pernix K1 and S. acidocaldarius DSM 639 were highly correlated with the gene expression level, but no such association was detected in P. aerophilum str. IM2 genomes.

Objectives:Molecular margin analysis is considered more sensitive in detecting preneoplastic lesions and residual cancer cells than conventional histological margin examination. Hence, we examined MGMT expression profile and methylation status in histologically negative margins of colorectal cancer patients.Design and methods:This study included 24 colorectal tumor tissues and corresponding negative surgical margin tissues. MGMT promoter methylation patterns were analyzed by using methylation-specific oligonucleotide microarray. In addition, MGMT protein expression was analyzed by immunohistochemistry. MGMT clinical significance was evaluated together with other well-known clinicopathological factors.Results:Extensive MGMT promoter methylation was observed in tumor tissues; a moderate methylation level was found in surgical margin tissues and little or no methylation was observed in the normal control. There was a trend towards longer overall survival for those patients with negative MGMT immunostaining in surgical margins.Conclusions:MGMT expression negative in surgical margin tissues indicates longer overall survival for colorectal tumor patients.

PurposeAutism spectrum disorder (ASD) is a neurodevelopmental disorder, of which Asperger syndrome and high-functioning autism are subtypes. Our goal is: 1) to determine whether a diagnostic model based on single-nucleotide polymorphisms (SNPs), brain regional thickness measurements, or brain regional volume measurements can distinguish Asperger syndrome from high-functioning autism; and 2) to compare the SNP, thickness, and volume-based diagnostic models.Material and MethodsOur study included 18 children with ASD: 13 subjects with high-functioning autism and 5 subjects with Asperger syndrome. For each child, we obtained 25 SNPs for 8 ASD-related genes; we also computed regional cortical thicknesses and volumes for 66 brain structures, based on structural magnetic resonance (MR) examination. To generate diagnostic models, we employed five machine-learning techniques: decision stump, alternating decision trees, multi-class alternating decision trees, logistic model trees, and support vector machines.ResultsFor SNP-based classification, three decision-tree-based models performed better than the other two machine-learning models. The performance metrics for three decision-tree-based models were similar: decision stump was modestly better than the other two methods, with accuracy = 90%, sensitivity = 0.95 and specificity = 0.75. All thickness and volume-based diagnostic models performed poorly. The SNP-based diagnostic models were superior to those based on thickness and volume. For SNP-based classification, rs878960 in GABRB3 (gamma-aminobutyric acid A receptor, beta 3) was selected by all tree-based models.ConclusionOur analysis demonstrated that SNP-based classification was more accurate than morphometry-based classification in ASD subtype classification. Also, we found that one SNP—rs878960 in GABRB3—distinguishes Asperger syndrome from high-functioning autism.

PurposeAutism spectrum disorder (ASD) is a neurodevelopmental disorder, of which Asperger syndrome and high-functioning autism are subtypes. Our goal is: 1) to determine whether a diagnostic model based on single-nucleotide polymorphisms (SNPs), brain regional thickness measurements, or brain regional volume measurements can distinguish Asperger syndrome from high-functioning autism; and 2) to compare the SNP, thickness, and volume-based diagnostic models.Material and MethodsOur study included 18 children with ASD: 13 subjects with high-functioning autism and 5 subjects with Asperger syndrome. For each child, we obtained 25 SNPs for 8 ASD-related genes; we also computed regional cortical thicknesses and volumes for 66 brain structures, based on structural magnetic resonance (MR) examination. To generate diagnostic models, we employed five machine-learning techniques: decision stump, alternating decision trees, multi-class alternating decision trees, logistic model trees, and support vector machines.ResultsFor SNP-based classification, three decision-tree-based models performed better than the other two machine-learning models. The performance metrics for three decision-tree-based models were similar: decision stump was modestly better than the other two methods, with accuracy = 90%, sensitivity = 0.95 and specificity = 0.75. All thickness and volume-based diagnostic models performed poorly. The SNP-based diagnostic models were superior to those based on thickness and volume. For SNP-based classification, rs878960 in GABRB3 (gamma-aminobutyric acid A receptor, beta 3) was selected by all tree-based models.ConclusionOur analysis demonstrated that SNP-based classification was more accurate than morphometry-based classification in ASD subtype classification. Also, we found that one SNP—rs878960 in GABRB3—distinguishes Asperger syndrome from high-functioning autism.

Background: Sex differences in cognitive tasks have been widely investigated. With brain-imaging techniques, the functions of the brain during the performance of tasks can be examined.Objective: Mental arithmetic and near-infrared spectroscopy (NIRS) were used to assess sex differences in prefrontal area activation in a functional brain study.Methods: Healthy college students were recruited to perform 2 mental arithmetic tasks. In the first (easy) task, students had to subtract a 1-digit number from a 3-digit number. In the second (difficult) task, they had to subtract a 2-digit number from a 3-digit number. Changes in the concentration of oxygenated hemoglobin (oxy-Hgb) in the prefrontal area during the tasks were measured with NIRS.Results: Thirty students (15 men, 15 women; mean [SD] age: 24.9 [2.2] and 24.3 [2.6] years, respectively) were recruited from Southeast University, Nanjing, China, to participate in the study. The concentration of oxy-Hgb increased during both mental arithmetic tasks (difficult task vs easy task, mean [SD] % arbitrary units: 4.36 [4.38] vs 2.26 [2.82]; F1,28 = 222.80; P < 0.01). Significant interactions of task x sex (F1,28 = 82.95), time × sex (F1,28 = 34.48), task × time (F1,28 = 222.57), and task × time × sex (F1,28 = 83.09) were obtained (all, P < 0.01). However, for the 2 tasks, no significant differences between men and women were observed in the mean (SD) response time (men vs women, sec: 3.60 [0.74] vs 3.56 [0.49] for the easy task, 6.55 [0.77] vs 6.44 [0.75] for the difficult task; F1,28 = 0.67; P = NS) or accuracy rate (men vs women, %: 95.33 [7.40] vs 92.77 [8.80] for the easy task, 62.67 [28.56] vs 54.67 [18.75] for the difficult task; F1,28 = 0.54; P = NS). Male students showed neural efficiency (less prefrontal activation in subjects with better performance) during the difficult task.Conclusions: In these subjects, sex differences in prefrontal response when performing mental arithmetic were associated with the intensity of the task. Compared with men, women had greater efficiency in task performance (ie, less activation or oxygen consumption for equal performance).

•A method to measure the proper region for microbial community reconstruction.•The preferential genus-specific amplicons are not always located in HVRs.•Low-abundance genera should may be ignored by “universal” primers.•Using multiple regions can improve the microbial community reconstruction.Background16S rRNA genes have been widely used for phylogenetic reconstruction and the quantification of microbial diversity through the application of next-generation sequencing technology. However, long-read sequencing is still costly, while short-read sequencing carries less information for complex microbial community profiling; therefore, the applications of high throughput sequencing platforms still remain challenging in microbial community reconstruction analysis.ResultsHere, we developed a method to investigate the profile of aligned 16S rRNA gene sequences and to measure the proper region for microbial community reconstruction, as a step in creating a more efficient way to detect microorganism at the genus level. Finally, we found that each genus has its own preferential genus-specific amplicons for a genus assignment, which are not always located in hyper variable regions (HVRs). It was also noted that the rare genera should contribute less than dominant ones to the common profile of the aligned 16S rRNA sequences and have lower affinity to the common universal primer.ConclusionsTherefore, using multiple 16S rRNA regions rather than one “universal” region can significantly improve the ability of microbial community reconstruction. In addition, we found that a short fragment is suitable for most genera identifications, and the proper conserved regions used for primer design are larger than before.

Determining the binding sites of the transcription factor is important for understanding of transcriptional regulation. Transcription factor c-Jun plays an important role in cell growth, differentiation and development, but the binding sites and the target genes are not clearly defined in the whole human genome. In this study, we performed a ChIP-Seq experiment to identify c-Jun binding site in the human genome. Forty-eight binding sites were selected to process further evaluation by dsDNA microarray assay. We identified 283 c-Jun binding sites in K562 cells. Data analysis showed that 48.8% binding sites located within 100 kb of the upstream of the annotated genes, 28.6% binding sites comprised consensus TRE/CRE motif (5′-TGAC/GTCA-3′, 5′-TGACGTCA-3′) and variant sequences. Forty-two out of the selected 48 binding sites were found to bind the c-Jun homodimer in dsDNA microarray analysis. Data analysis also showed that 1569 genes are located in the neighborhood of the 283 binding sites and 191 genes in the neighborhood of the 42 binding sites validated by dsDNA microarray. We consulted 38 c-Jun target genes in previous studies and 16 among these 38 genes were also detected in this study. The identification of c-Jun binding sites and potential target genes in the genome scale may improve our fundamental understanding in the molecular mechanisms underlying the transcription regulation related to c-Jun.