Detection of Genetically Modified Crops by Combination of Multiplex PCR and Low-density DNA Microarray

Objective To develop a technique for simultaneous detection of various target genes in Roundup Ready soybean by combining multiplex PCR and low-density DNA microarray. Methods Two sets of the multiplex PCR system were used to amplify the target genes in genetically modified （GM） soybean. Seventeen capture probes （PCR products） and 17 pairs of corresponding primers were designed according to the genetic characteristics of Rroundup Ready soybean （GTS40-3-2）, maize （MonS10, Nk603, GA21）, canola （T45, MS1/RF1）, and rice （SCK） in many identified GM crops. All of the probes were categorized and identified as species-specific probes. One negative probe and one positive control probe were used to assess the efficiency of all reactions, and therefore eliminate any false positive and negative results. After multiplex PCR reaction, amplicons were adulterated with Cy5-dUTP and hybridized with DNA microarray. The array was then scanned to display the specific hybridization signals of target genes. The assay was applied to the analysis of sample of certified transgenic soybean （Roundup Ready GTS40-3-2） and canola （MS1/RF1）. Results A combination technique of multiplex PCR and DNA microarray was successfully developed to identify multi-target genes in Roundup Ready soybean and MS 1/RF1 canola with a great specificity and reliability. Reliable identification of genetic characteristics of Roundup Ready of GM soybean from genetically modified crops was achieved at 0.5% transgenic events, indicating a high sensitivity. Conclusion A combination technique of multiplex PCR and low-density DNA microarray can reliably detect and identify the genetically modified crops.

Controlling DNA Bundle Size and Spatial Arrangement in Selfassembled Arrays on Superhydrophobic Surface

The use of superhydrophobic surfaces(SHSs) is now emerging as an attractive platform for the realization of one-dimensional(1D) nanostructures with potential applications in many nanotechnological and biotechnological fields.To this purpose, a strict control of the nanostructures size and their spatial arrangement is highly required. However, these parameters may be strongly dependent on the complex evaporation dynamics of the sessile droplet on the SHS. In this work, we investigated the effect of the evaporation dynamics on the size and the spatial arrangement of self-assembled 1D DNA bundles. Our results reveal that different arrangements and bundle size distributions may occur depending on droplet evaporation stage. These results contribute to elucidate the formation mechanism of 1D nanostructures on SHSs.

TCERG1L hypermethylation is a risk factor of diabetic retinopathy in Chinese children with type 1 diabetes

●AIM:To identify the differential methylation sites(DMS)and their according genes associated with diabetic retinopathy(DR)development in type 1 diabetes(T1DM)children.●METHODS:This study consists of two surveys.A total of 40 T1DM children was included in the first survey.Because no participant has DR,retina thinning was used as a surrogate indicator for DR.The lowest 25%participants with the thinnest macular retinal thickness were included into the case group,and the others were controls.The DNA methylation status was assessed by the Illumina methylation 850K array BeadChip assay,and compared between the case and control groups.Four DMS with a potential role in diabetes were identified.The second survey included 27 T1DM children,among which four had DR.The methylation patterns of the four DMS identified by 850K were compared between participants with and without DR by pyrosequencing.●RESULTS:In the first survey,the 850K array revealed 751 sites significantly and differentially methylated in the case group comparing with the controls(|Δβ|>0.1 and Adj.P<0.05),and 328 of these were identified with a significance of Adj.P<0.01.Among these,319 CpG sites were hypermethylated and 432 were hypomethylated in the case group relative to the controls.Pyrosequencing revealed that the transcription elongation regulator 1 like(TCERG1L,cg07684215)gene was hypermethylated in the four T1DM children with DR(P=0.018),which was consistent with the result from the first survey.The methylation status of the other three DMS(cg26389052,cg25192647,and cg05413694)showed no difference(all P>0.05)between participants with and without DR.●CONCLUSION:The hypermethylation of the TCERG1L gene is a risk factor for DR development in Chinese children with T1DM.

Precise Microdeletion Detection of Prader-Willi Syndrome with Array Comparative Genome Hybridization

Objective Prader-Willi Sydrome （PWS） is a human disorder related to genomic imprinting defect on 15ql 1-13. It is characterized by a series of classic features such as hypotonia, hyperphagia, obesity, osteoporosis, typical facial and body dysmorphosis, hypogonadism, mental and behaviour disorders. Our study was designed to precisely detect the microdeletions, which accounts for 65%-70% of the PWS. Methods Physical and laboratory examinations were firstly performed to diagnose PWS clinically, and to discover novel clinical features. Then the patient was screened with bisulfite-specific sequencing and precisely delineated through high-density array CGH. Results With the bisulfite-specific sequencing, the detected CpG island in the PWS critical region was found homozygously hypermethylated. Then with array CGH, a 2.22 Mb type II microdeletion was detected, covering a region from MKRN3, MAGEL2, NDN, PWRN2, PWRN1, Cl2orf2, SNURF-SNRPN, C/D snoRNAs, to distal of UBE3A. Conclusions Array CGH, after the fast screening of Bisulfite-specific sequencing, is a feasible and precise method to detect microdeletions in PWS patients. A novel feature of metacarpophalangeal joint rigidity was also presented, which is the first time reported in PWS.

Microarray,SAGE and their applications to cardiovascular diseases

The wealth of DNA data generated by the human genome project coupling with recently invented high-throughput gene expression profiling techniques has dramatically sped up the process for biomedical researchers on elucidating the role of genes in human diseases. One powerful method to reveal insight into gene functions is the systematic analysis of gene expression. Two popular high-throughput gene expression technologies, microarray and Serial Analysis of Gene Expression (SAGE) are capable of producing large amounts of gene expression data with the potential of providing novel insights into fundamental disease processes, especially complex syndromes such as cardiovascular disease, whose etiologies are due to multiple genetic factors and their interplay with the environment. Microarray and SAGE have already been used to examine gene expression patterns of cell-culture, animal and human tissues models of cardiovascular diseases. In this review, we will first give a brief introduction of microarray and SAGE technologies and point out their limitations. We will then discuss the major discoveries and the new biological insightsthat have emerged from their applications to cardiovascular diseases. Finally we will touch upon potential challenges and future developments in this area.

Spinal deformity measurement using a low-density flexible array ultrasound transducer:A feasibility study with phantoms

Spinal deformities assessment using 3D ultrasound scanning has limitations in fitting onto different back surface contour as well as fitting within the gaps between subject and their spinal brace during bracing assessments.The study proposed a flexible array ultrasound transducer to overcome these limitations.The results demonstrated the feasibility of spinal deformity assessments with a flexible ultrasound array when arranged in four shapes,namely Linear,Concave,Convex,and S-shaped.For comparisons of imaging performance on spinous process using the four shapes,Convex and S-shaped transducer showed a depth dependence and lateral location dependence of the lateral intensity distribution of spinous process,respectively.S-shaped transducer had the least accurate prediction of the location of spinous process,with measurement error of 4.8
3.2 mm,it also showed poorer prediction on spinal curvature measurements.This is suggested to be due to the asymmetrical distortion to the spinous process due to the lateral location dependence of the image.However,the coronal curve prediction of spine phantom performed well with R-squared values of>0.97 in all transducer shapes.The results of this study paved the way for further investigation on the improvement of image quality and measurement accuracy under different shapes for the flexible array,mechanism of dynamic shape change during the scanning to fit different body contour,as well as extension from 1D to 2D flexible array.

Implementation of encoder and decoder for LDPC codes based on FPGA

This paper proposes a parallel cyclic shift structure of address decoder to realize a high-throughput encoding and decoding method for irregular-quasi-cyclic low-density parity-check(IR-QC-LDPC)codes,with a dual-diagonal parity structure.A normalized min-sum algorithm(NMSA)is employed for decoding.The whole verification of the encoding and decoding algorithm is simulated with Matlab,and the code rates of 5/6 and 2/3 are selected respectively for the initial bit error ratio as 6%and 1.04%.Based on the results of simulation,multi-code rates are compatible with different basis matrices.Then the simulated algorithms of encoder and decoder are migrated and implemented on the field programmable gate array(FPGA).The 183.36 Mbps throughput of encoder and the average 27.85 Mbps decoding throughput with the initial bit error ratio 6%are realized based on FPGA.

Analysis of low-density lipoprotein receptor gene mutations in a Chinese patient with clinically homozygous familial hypercholesterolemia

Objective To screen the point mutation of the low-density lipoprotein receptor (LDL-R) gene in Chinese familial hypercholesterolemia (FH) patients,characterize the relationship between the genotype and the phenotype and discuss the molecular pathological mechanism of FH. Methods A patient with clinical phenotype of homozygous FH and her parents were investigated for mutations in the promoter and all eighteen exons of the LDL-R gene. Screening was carried out using Touch-down PCR and direct DNA sequencing; multiple alignment analysis by DNASIS 2.5 was used to find base alteration,and the LDL-R gene mutation database was searched to identify the alteration. In addition,the apolipoprotein B gene (apo B) was screened for known mutations (R3500Q) that cause familial defective apo B 100 (FDB) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).Results Two new heterozygous mutations in exons 4 and 9 of the LDL-R gene were identified in the proband (C122Y and T383I) as well as her parents. Both of the mutations have not been published in the LDL-R gene mutation database. No mutation of apo B 100 (R3500Q) was observed. Conclusion Two new mutations (C112Y and T383I) were found in the LDL-R gene,which may result in FH and may be particularly pathogenetic genotypes in Chinese people.

Diversity Suppression-Subtractive Hybridization Array for Profiling Genomic DNA Polymorphisms

Genomlc DNA polymorphlsms are very useful for tracing genetic traits end studying biological diversity among species. Here, we present a method we call the ＂diversity suppresslon-subtractlve hybridization array＂ for effectively profiling genomlc DNA polymorphisms. The method first obtains the subtracted gDNA fragments between any two species by suppression subtraction hybridization （SSH） to establish e subtracted gDNA library, from which diversity SSH arrays are created with the selected subtracted clones. The diversity SSH array hybridizes with the DIG-labeled genomlc DNA of the organism to be assayed. Six closely related Dendrobium species were studied as model samples. Four Dendrobium species as testers were used to perform SSH. A total of 617 subtracted positive clones were obtained from four Dendrobium species, and the average ratio of positive clones was 80.3%. We demonstrated that the average percentage of polymorphlc fragments of palrwlse comparisons of four Dendrobium species was up to 42.4%. A dendrogram of the relatedness of six Dendrobium species was produced according to their polymorphic profiles. The results revealed that the diversity SSH array Is a highly effective platform for profiling genomlc DNA polymorphlsms and dendrograms.

Disposable paper-based bipolar electrode array for multiplexed electrochemiluminescence detection of pathogenic DNAs

A novel disposable paper-based bipolar electrode （BPE） array is fabricated for multiplexed electrochemiluminescence （ECL） detection of pathogenic DNAs. This proposed BPE array device consists of 15 units, each consisting of six sensing cells and two reporting cells patterned using hydrophobic wax. A hairpin structure DNA assembled on the cathodes of BPEs hybridizes with Pt nanoparticles （NPs） labeled probe DNA in the presence of complementary target DNA. The introduction of Pt NPs catalyzes the reduction of dissolved 02 at cathodes and induces an enhanced ECL signal from Ru（bpy）32＋/tripropylamine （TPrA） at the anodes of BPEs. The dissolved 02 lost in reduction reaction could be promptly replenished due to the relatively large contact area of the paper-based cells with air, which ensures the stability of ECL signal. This obtained paper-based BPE array sensor showed excellent performances for the multiplexed analysis of the syphilis （Treponema pallidum） gene, the immunodeficiency virus gene （HIV） and hepatitis B virus gene （HBV）.

Design and implementation of LDPC encoder based on FPGA

A low density parity check(LDPC)encoder with the codes of(8176,7154)and encoding rate of 7/8 under CCSDS standard for near space communication is designed.Based on LDPC encoding theory,the FPGA-based coding algorithm is designed.Based on the characteristics of LDPC generating matrix,the cyclic shift register is introduced as the core of the encoding circuit,and the shift-register-Adder-Accumulator(SRAA)structure is adopted to realize the fast calculation of matrix multiplication,so as to construct the encoding module with partial parallel encoding circuit as the core.In addition,the serial port input and output module,RAM storage module and control module are also designed,which together constitute the encoder system.The design scheme is implemented by FPGA hardware and verified by simulation and experiment.The results show that the test results of the designed LDPC encoder are consistent with the theoretical results.Therefore,the coding system is practical,and the design method is simple and efficient.

A new method of preparing fiber-optic DNA biosensor and its array for gene detection

A new method of preparing fiber-optic DNA biosensor and its arrayfor the simultaneous detection of multiple genes is described. The optical fibers were first treated with poly-l-lysine, and then were made into fiber-optic DNA biosensors by adsorbing and immobilizing the oligonucleotide probe on its end. By assembling the fiber-optic DNA biosensors in a bundle in which each fiber carried a different DNA probe, the fiber-optic DNA biosensor array was well prepared. Hybridization of fluorescent- labeled cDNA of p53 gene, N-ras gene and Rb1 gene to the DNA array was monitored by CCD camera. A good result was achieved.

Express and sensitive detection of multiple miRNAs via DNA cascade reactors functionalized photonic crystal array

Array based detection techniques with fluorescence signal reading is a powerful tool for multiple targets analysis. However,when applied fluorescence array for micro RNA detection, time-consuming multi-steps surface signal amplification is usually required due to the low abundance of micro RNA in total RNA expressions, which impairs detection efficiency and limits its application in point of care test(POCT) manner. Herein, DNA cascade reactors(DCRs) functionalized photonic crystal(PC)array was fabricated for express and sensitive detections of mi RNA-21 and mi RNA-155. DCRs were assembled by interval conjugation of self-quenched hairpin DNA probes to single strand DNA nanowire synthesized by rolling circle amplification,which generated cascade DNA hybridization reactions in response to target mi RNAwith instant fluorescence recovery signal. PC array patterns with multi-structure colors further amplified fluorescence with their respective photonic bandgaps(PBGs)matching with the emission peaks of fluorescence molecules labelled on DCRs. The as-prepared DCRs functionalized PC array demonstrated express and sensitive simultaneous detections of mi RNA-21 and mi RNA-155 with hundreds f M detection limits only in 15 min, and was successfully applied in fast quantifications of low abundance mi RNAs from cell lysates and spiked mi RNAs from human serum, which would hold great potential for disease diagnosis and therapeutic effect monitoring with a POCT manner.

Genetic Copy Number Variations in Colon Mucosa Indicating Risk for Colorectal Cancer

Background: Sporadic colorectal tumors probably carry genetic alterations that may be related to familiar clusters according to risk loci visualized by SNP arrays on normal tissues. The aim of the present study was therefore to search for DNA regions (copy number variations, CNVs) as biomarkers associated to genetic susceptibility for early risk predictions of colorectal cancer. Such sequence alterations could provide additional information on phenotypic grouping of patients. Material and Methods: High resolution 105K oligonucleotide microarrays were used in search for CNV loci in DNA from tumor-free colon mucosa at primary operations for colon cancer in 60 unselected patients in comparison to DNA in buffy coat cells from 44 confirmed tumor-free and healthy blood donors. Array-detected CNVs were confirmed by Multiplex ligation-dependent probe amplification (MLPA). Results: A total number of 205 potential CNVs were present in DNA from colon mucosa. 184 (90%) of the 205 potential CNVs had been identified earlier in mucosa DNA from healthy individuals as reported to the Database of Genomic Variants. Remaining 21 (10%) CNVs were potentially novel sites. Two CNVs (3q23 and 10q21.1) were significantly related to colon cancer, but not confirmed in buffy coat DNA from the cancer patients. Conclusion: Our study reveals two CNVs that indicate increased risk for colon cancer;These DNA alterations may have? been acquired by colon stem cells with subsequent appearance among epithelial mucosa cells. Impact: Certain mucosa CNV alterations may indicate individual susceptibility for malignant transformation in relationship to intestinal toxins and bacterial growth.

LP-LDPC:Three-Level Parallel FPGA Architecture for Fast Prototyping of LDPC Decoder Using High-Level Synthesis

Low-Density Parity-heck Codes(LDPC)with excellent error-correction capabilities have been widely used in both data communication and storage fields,to construct reliable cyber-physical systems that are resilient to real-world noises.Fast prototyping field-programmable gate array(FPGA)-based decoder is essential to achieve high decoding performance while accelerating the development process.This paper proposes a three-level parallel architecture,TLP-LDPC,to achieve high throughput by fully exploiting the characteristics of both LDPC and underlying hardware while effectively scaling to large-size FPGA platforms.The three-level parallel architecture contains a low-level decoding unit,a mid-level multi-unit decoding core,and a high-level multi-core decoder.The low-level decoding unit is a basic LDPC computation component that effectively combines the features of the LDPC algorithm and hardware with the specific structure(e.g.,Look-Up-Table,LUT)of the FPGA and eliminates potential data conflicts.The mid-level decoding core integrates the input/output and multiple decoding units in a well-balancing pipelined fashion.The top-level multi-core architecture conveniently makes full use of board-level resources to improve the overall throughput.We develop an LDPC C++code with dedicated pragmas and leverage HLS tools to implement the TLP-LDPC architecture.Experimental results show that TLP-LDPC achieves 9.63 Gbps end-to-end decoding throughput on a Xilinx Alveo U50 platform,3.9x higher than existing HLS-based FPGA implementations.

On-chip detection of a single nucleotide polymorphism without polymerase amplification

A nanoparticle-assembled photonic crystal （PC） array was used to detect single nucleotide polymorphism （SNP）. The assay platform with PC nanostructure enhanced the fluorescent signal from nanoparticle-hybridized DNA complexes due to phase matching of excitation and emission. Nanoparticles coupled with probe DNA were trapped into nanowells in an array by using an electrophoretic particle entrapment system. The PC/DNA assay platform was able to identify a 1 base pair （bp） difference in synthesized nucleotide sequences that mimicked the mutation seen in a feline model of human autosomal dominant polycystic kidney disease （PKD） with a sensitivity of 0.9 fg/mL （50 aM）-sensitivity, which corresponds to 30 oligos/array. The reliability of the PC/DNA assay platform to detect SNP in a real sample was demonstrated by using genomic DNA （gDNA） extracted from the urine and blood of two PKD-wild type and three PKD positive cats. The standard curves for PKD positive （PKD＋） and negative （PKD-） DNA were created using two feline-urine samples. An additional three urine samples were analyzed in a similar fashion and showed satisfactory agreement with the standard curve, confirming the presence of the mutation in affected urine. The limit of detection （LOD） was 0.005 ng/mL which corresponds to 6 fg per array for gDNA in urine and blood. The PC system demonstrated the ability to detect a number of genome equivalents for the PKD SNP that was very similar to the results reported with real time polymerase chain reaction （PCR）. The favorable comparison with quantitative PCR suggests that the PC technology may find application well beyond the detection of the PKD SNP, into areas where a simple, cheap and portable nucleic acid analvsis is desirable.

Novel diagnosis and therapy for hepatoma targeting HBV-related carcinogenesis through alternative splicing of FIR (PUF60)/FIRΔexon2

Aim: Disturbed alternative splicing of far upstream element-binding protein-interacting repressor (FIR) was found to be unable to repress c-Myc transcription and so it might be important for suppressing tumor development. FIR is a splicing variant of poly (U)-binding-splicing factor (PUF60), and forms complex with other splicing factors. FIR/PUF60 is a splicing factor of U2 small nuclear ribonucleoprotein auxiliary factor family, Thus FIR/PUF60 is a multifunctional protein. The expression of exon2-lacking splicing variant of FIR, FIRΔexon2, is elevated in many cancer tissues and promotes tumor development by disabling FIR-repression to sustain c-Myc activation. FIRΔexon2, as a dominant negative of FIR, opposed apoptosis in cancer cells. FIR/FIRΔexon2 interacts with degron pocket of F-box and W (Typ) D (Asp) repeat domain-containing 7 and inhibits proteolysis of substrates proteins. Recently, FIR/PUF60 was identified as a versatile regulator of transcriptional and post-transcriptional steps in expression of hepatitis B virus (HBV) pregenomic RNA (pgRNA) expression. Methods: Small molecular chemical compounds against FIR and FIRΔexon2 were screened among 2,3275 chemicals by natural product depository array (RIKEN, Wako, Saitama, Japan). Results: Nine chemicals against FIR and four chemicals against FIRΔexon2 were identified as candidates of interacting chemicals. Interestingly, BK697 contains WD -like structure. Among them, BK697 against FIRΔexon2 inhibited hepatoma cell growth. Conclusion: Therefore, FIR (PUF60)/FIRΔexon2 is multifunctional and applicable for clinical use for HBV suppression and hepatoma treatment. Together, one clue to the development of hepatome diagnosis and therapies directed against FIR/FIRΔexon2/PUF60 with small molecular weight chemicals that inhibit HBV cccDNA replication.