PCR-SSCP-DNA sequencing method in detecting PTEN gene mutation and its signifi cance in human gastric cancer

AIM: To discuss the possible effect of PTEN gene mutations on occurrence and development of gastric cancer. METHODS: Fifty-three gastric cancer specimens were selected to probe PTEN gene mutations in genome of gastric cancer and paracancerous tissues using PCR-SSCP-DNA sequencing method based on microdissection and to observe the protein expression by immunohistochemistry technique. RESULTS: PCR-SSCP-DNA sequencing indicated that 4 kinds of mutation sites were found in 5 of 53 gastric cancer specimens. One kind of mutation was found in exons. AA-TCC mutation was located at 40bp upstream of 3’ lateral exon 7 (115946 AA-TCC). Such mutations led to terminator formation in the 297th codon of the PTEN gene. The other 3 kinds of mutation were found in introns,including a G-C point mutation at 91 bp upstream of 5’ lateral exon 5(90896 G-C),a T-G point mutation at 24 bp upstream of 5’ lateral exon 5 (90963 T-G),and a single base A mutation at 7 bp upstream of 5’ lateral exon 5 (90980 A del). The PTEN protein expression in gastric cancer and paracancerous tissues detected using immunohistochemistry technique indicated that the total positive rate of PTEN protein expression was 66% in gastric cancer tissue,which was significantly lower than that (100%) in paracancerous tissues (P < 0.005). CONCLUSION: PTEN gene mutation and expression may play an important role in the occurrence and development of gastric cancer.

Complementary DNA sequencing (cDNA): an eff ective approach for assessing the diversity and distribution of marine benthic ciliates along hydrographic gradients

The Yellow Sea Cold Water Mass(YSCWM)is a distinct hydrographic phenomenon of the Yellow Sea,and the distribution pattern of meio-and macrobenthos diff ers inside and outside of the YSCWM.However,such a pattern has never been observed in the microbenthic ciliate communities.Therefore,we hypothesized that benthic ciliates followed a similar distribution pattern as meio-and macrobenthos,but this pattern has not been uncovered by morphological methods.We evaluated the diversity and distribution of benthic ciliates at fi ve stations along hydrographic gradients across the YSCWM and adjacent shallow water by using morphology and DNA and complementary DNA(cDNA)high-throughput sequencing of the V4 region of 18S rRNA gene.Results showed that the diversity of benthic ciliates detected by DNA(303 OTUs),and the cDNA(611 OTUs)sequencing was much higher than that detected by the morphological method(79 species).Morphological method detected roughly diff erent ciliate communities inside and outside of the YSCWM,but without statistical signifi cance.No clear pattern was obtained by DNA sequencing.In contrast,cDNA sequencing revealed a distinct distribution pattern of benthic ciliate communities like meioand macrobenthos,which coincided well with the results of the environmental parameter analysis.More than half of the total sequences detected by DNA sequencing belonged to planktonic ciliates,most(if not all)of which were recovered from historic DNA originating through the sedimentation of pelagic forms because none of them were observed morphologically.The irrelevant historic DNA greatly infl uenced the recovery of rare species and thus limited the understanding of the benthic ciliate diversity and distribution.Our research indicates that the methods used have signifi cant eff ects on the investigation of benthic ciliate communities and highlights that cDNA sequencing has great advantages in estimating the diversity and distribution of benthic ciliates,as well as the potential for benthic environmental assessments.

A New Method of Identification on Edible Lycium Linn. Germplasm Resources——nrDNA ITS Sequencing

[ Objective ] The study aimed to identify Lycium Linn. at molecular level. [ Method ] The nrDNA ITS sequence of 5 edible Lycium Linn. germplasm resources were investigated. [ Result ] The nrDNA ITS regions of five edible Lycium Liun. germplasm resources were sequenced. The whole sequences varied from 628 bp to 632 bp, with the average length of 630 bp. Total 79 variation sites were observed in the sequences, which accounts for 12. 5%. [ Conclusion] Sequence analysis based on nrDNA sequencing provides a new approach to identify edible Lycium Linn. germplasm resources.

Preliminary Studies on Identification of Lycium Linn.Germplasm Resources by nrDNA ITS Sequencing

[Objective] The study aimed to identify woltberry （Lycium Linn.） germplasm resources at molecular level by analyzing the nrDNA ITS sequence. [Method] Genomic DNA from woltberry leaves extracted by modified CTAB method were regarded as templates for PCR amplification by specific primer, clone and sequencing. [Result] The nrDNA ITS sequences were obtained and then differentiated among three tested materials. [Conclusion] PCR amplification and sequencing on nrDNA ITS is a feasible approach to identify different woltberry germplasm resources.

DNA sequencing by synthesis with degenerate primers

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.

DNA Sequencing by Capillary Electrophoresis Using Quasi-inter-penetrating Network Formed by Polyacrylamide and Poly(N-hydroxymethylacrylamide)

Quasi-interpenetrating network formed by polyacrylamide and poly （N-hydroxymethylacrylamide） was designed, synthesized, and tested for DNA sequencing by capillary electrophoresis. The performance of quasi-IPN on DNA sequencing was determined by the acrylamide to N-hydroxymethylacrylamide molar ratio and sequencing temperature.

Genome Sequencing Using Graph Theory Approach

Genome sequencing is the process of determining in which order the nitrogenous bases also known as nucleotides within a DNA molecule are arranged. Every organism’s genome consists of a unique sequence of nucleotides. These nucleotides bases provide the phenotypes and genotypes of a cell. In mathematics, Graph theory is the study of mathematical objects known as graphs which are made of vertices (or nodes) connected by either directed edges or indirect edges. Determining the sequence in which these nucleotides are bonded can help scientists and researchers to compare DNA between organisms, which can help show how the organisms are related. In this research, we study how graph theory plays a vital part in genome sequencing and different types of graphs used during DNA sequencing. We are going to propose several ways graph theory is used to sequence the genome. We are as well, going to explore how the graphs like Hamiltonian graph, Euler graph, and de Bruijn graphs are used to sequence the genome and advantages and disadvantages associated with each graph.

Microdissection of Haynaldia villosa Telosome 6VS and Cloning of Species-specific DNA Sequences

The material T240_6 derived from SC 2 young embryo of the combination CA9211/RW15 (6D/6V alien substitution) was telosomic substitution line of 6VS identified by GISH (genomic in situ hybridization) analysis. The 6VS was microdissected with a needle and transferred into a 0.5 mL Ep tube. In the 'single tube', all the subsequence steps were conducted. After two round of LA (Linker adaptor)_PCR amplification, the size of PCR bands ranged from 100 to 3 000 bp, with predominate bands 600-1 500 bp. The products were confirmed by Southern blotting analysis using Haynaldia villosa (L.) Schur. genomic DNA labeled with 32 P as probe. The PCR products were purified and ligated into clone vector-pGEM_T easy vector. Then, the plasmids were transformed into competence E. coli JM109 with cool CaCl 2. It was estimated that there were more than 17 000 white clones in the library. The size of insert fragments distributed from 100-1 500 bp, with average of 600 bp. Using H. villosa genomic DNA as probe, dot blotting results showed that 37% clones displayed strong and medium positive signals, and 63% clones had faint or no signals. It is demonstrated that there were about 37% repeat sequence clones and 67% single/unique sequence clones in the library. Eight H. villosa_specific clones were screened from the library, and two clones pHVMK22 and pHVMK134 were used for RFLP analysis and sequencing. Both of them were H. villosa specific clones. The pHVMK22 was a unique sequence clone, and the pHVMK134 was a repeat sequence clone. When the pHVMK22 was used as a probe for Southern hybridization, all the powdery mildew resistance materials showed a special band of 2 kb, while all the susceptible ones not. The pHVMK22 may be applied to detect the existence of Pm21.

Isolation of Zizania latifolia Species-specific DNA Sequences and Their Utility in Identification of Z. latifolia DNA Introgressed into Rice

根据两个植物抗病基因N和RPS2中核酸结合位点 (NBS)和富亮氨酸重复区 (LRR)中的保守序列设计了一对特异引物 ,用PCR从具有水稻 (OryzasativaL .)改良所需要的许多优良性状的水稻近缘野生种菰 (Zizanialatifolia(Griseb .)Turcz.exStapf)的基因组DNA中扩增同源片段。PCR产物经克隆后 ,分别以菰和水稻的基因组DNA为探针 ,通过点杂交对所得克隆进行了分析。点杂交结果表明 ,在所分析的 6 0个克隆中有 2个克隆是菰专化的序列 ,即它们与水稻无杂交信号。基因组DNA的Southern杂交进一步证实了这 2个克隆的专化性。为了验证一些可能的“水稻_菰”渐渗杂交系是否确实含有源自供体菰的DNA ,以这 2个克隆为探针 ,与经EcoRⅠ酶切的 5个可能的渐渗杂交系进行了Southern杂交。结果表明 ,这 2个克隆均能检测出其中的一个系含有其同源序列。这一结果为曾经报道的经一种非常规有性杂交方法将菰DNA导入水稻提供了确凿的证据。

Characterization of Two Groups of Low_copy and Specific DNA Sequences Isolated from Chromosome 7B of Common Wheat

Recent work revealed that, in the genomes of polyploid wheat, there exists a class of low_copy and chromosome_specific sequences that are labile upon polyploid formation. This class of sequences was proposed to play a critical role in the stabilization and establishment of nascent plant polyploids as new species. To further study this issue, five wheat chromosome 7B_specific sequences, isolated from common wheat (Triticum aestivum L.) by chromosome microdissection, were characterized. The sequences were studied by genomic Southern hybridizations on a collection of polyploid wheats and their diploid progenitors. Four sequences hybridized to all polyploid species, but at the diploid level to only species closely related to the B_genome of polyploid wheat. This indicates that these sequences originated with the divergence of the diploid species, and was then vertically transmitted to polyploids. One sequence hybridized to all species at both the diploid and polyploid levels, suggesting its elimination after the polyploid wheat formation. The hybridization of this sequence to two synthetic polyploid wheats indicated that sequence elimination is a rapid event and probably related to methylation status of the sequence. Based on the above results, we suggest that selective changes of low_copy sequences occur rapidly after polyploid formation, which may contribute to the differentiation of chromosomes in newly formed allopolyploid wheats.

Discriminant Classification Model of DNA Sequence

[Objective] The research aimed to construct the discriminant classification model of DNA sequence by combining with the biology knowledge and the mathematical method.[Method] According to the polarity nature of side chain radical in the amino acid,the classification information of amino acid which represented the sequence characteristic from the content and array situation of base was extracted from the different sequences that the amino acid content was different.The four-dimension vector was used to represent.Mahalanobis distance and Fisher discriminant methods were used to classify the given sequence.[Result] In the model,the back substitution rates of sample obtained by two kinds of classification methods were both 100%,and the consistent rate of classification was 90%.[Conclusion] In the model,the calculation method was simple,and the accuracy of classification result was higher.It was superior to the discriminant classification model which was only based on the base content.

Phylogenetic, phylogeographic and divergence time analysis of Anopheles subpictus species complex using ITS2 and COI sequences

Objective:To address the phylogenetic and phylogeographic relationship between different lineages of Anopheles(An.)subpictus species complex in most parts of the Asian continent by maximum utilization of Internal Transcriber Spacer 2(ITS2)and cytochrome C oxidase I(COI)sequences deposited at the GenBank.Methods:Seventy-five ITS2,210 COI and 26 concatenated sequences available in the NCBI database were used.Phylogenetic analysis was performed using Bayesian likelihood trees,whereas median-joining haplotype networks and time-scale divergence trees were generated for phylogeographic analysis.Genetic diversity indices and genetic differentiation were also calculated.Results:Two genetically divergent molecular forms of An.subpictus species complex corresponding to sibling species A and B are established.Species A evolved around 37-82 million years ago in Sri Lanka,India,and the Netherlands,and species B evolved around 22-79 million years ago in Sri Lanka,India,and Myanmar.Vietnam,Thailand,and Cambodia have two molecular forms:one is phylogenetically similar to species B.Other forms differ from species A and B and evolved recently in the above mentioned countries,Indonesia and the Philippines.Genetic subdivision among Sri Lanka,India,and the Netherlands is almost absent.A substantial genetic differentiation was obtained for some populations due to isolation by large geographical distances.Genetic diversity indices reveal the presence of a long-established stable mosquito population,at mutation-drift equilibrium,regardless of population fluctuations.Conclusions:An.subpictus species complex consists of more than two genetically divergent molecular forms.Species A is highly divergent from the rest.Sri Lanka and India contain only species A and B.

p21^(WAF1/CIP1) Gene DNA Sequence Change and Their Relationship with the Phenotype of Human Osteosarcoma

Objective: To investigate the p21WAF1 /CIP1gene DNA sequence change and their relationship with the phenotype of human osteosarcoma. Methods: p21WAF1 /CIP1gene DNA of 36 osteosarcoma spec- imens was examined by using polymerase chain reaction-single strand conformation polymorphism (PCR- SSCP) method. The PCR products were sequenced directly. Results: In p21WAF1 /CIP1 gene exon3 of 36 cases of human osteosarcoma, the change of C→T in the p21WAF1 /CIP1gene CDNA sequence of position 609th occurred in 17 cases with the incidence being 44.4%. In 10 normal blood samples, DNA sequence analysis showed the change of C→T in the p21WAF1 /CIP1gene CDNA sequence of position 609th occurred in 8 cases with the incidence being 80%. Conclusion: The novel location of p21WAF1 /CIP1gene polymorphism of osteosarcoma, but not mutation was de?ned, and this location might provide the meaningful reference for the further research of p21WAF1/CIP1 gene.p2lWAF1/CIP1基因DNA序列分析及其与骨肉瘤表型的关系

Current blockade mechanism for DNA translocation through solid-state nanopore with different membrane thickness

The current blockade mechanism for λ -DNA translocation under electrical field is investigated through solid-state nanopores with different pore thicknesses. The conductance of a nanopore system mainly consists of the contribution of the pore and access region, and the latter becomes dominant when the nanopore thickness gradually decreases to atomic layer thickness. Based on the existing model of nanopore resistance, a simplified model which describes the relative current blockade during the X-DNA translocation through the nanopores is deduced to quantitatively present the relationship between nanopore thickness and relative current blockade. Results show that the relative current blockade is effectively increased by reducing the nanopore diameter but it decreases with the decreasing nanopore thickness. A two-stage schematic is proposed to increase the relative current blockade by setting a much smaller resistance region. Experimental results show a 21. 9% increase in the relative current blockade with the proposed schematic.

Molecular Taxonomy of Conogethes punctiferalis and Conogethes pinicolalis(Lepidoptera: Crambidae) Based on Mitochondrial DNA Sequences

Conogethes punctiferalis（Guenée）（Lepidoptera： Crambidae） was originally considered as one species with fruit-feeding type（FFT） and pinaceae-feeding type（PFT）, but it has subsequently been divided into two different species of Conogethes punctiferalis and Conogethes pinicolalis. The relationship between the two species was investigated by phylogenetic reconstruction using maximum-likelihood（ML） parameter estimations. The phylogenetic tree and network were constructed based upon sequence data from concatenation of three genes of mitochondrial cytochrome c oxidase subunits I, II and cytochrome b which were derived from 118 samples of C. punctiferalis and 24 samples of C. pinicolalis. The phylogenetic tree and network showed that conspecific sequences were clustering together despite intraspecific variability. Here we report the results of a combined analysis of mitochondrial DNA sequences from three genes and morphological data representing powerful evidence that C. pinicolalisand C. punctiferalis are significantly different.

Chaos game representation(CGR)-walk model for DNA sequences

Chaos game representation （CGR） is an iterative mapping technique that processes sequences of units, such as nucleotides in a DNA sequence or amino acids in a protein, in order to determine the coordinates of their positions in a continuous space. This distribution of positions has two features： one is unique, and the other is source sequence that can be recovered from the coordinates so that the distance between positions may serve as a measure of similarity between the corresponding sequences. A CGR-walk model is proposed based on CGR coordinates for the DNA sequences. The CGR coordinates are converted into a time series, and a long-memory ARFIMA （p, d, q） model, where ARFIMA stands for autoregressive fractionally integrated moving average, is introduced into the DNA sequence analysis. This model is applied to simulating real CGR-walk sequence data of ten genomic sequences. Remarkably long-range correlations are uncovered in the data, and the results from these models are reasonably fitted with those from the ARFIMA （p, d, q） model.

Comparison of Human Papillomavirus Detection and Genotyping with Four Different Prime Sets by PCR-Sequencing

Objective To assess and compare the Human Papillomavirus（HPV） detection efficiency and the potential clinical utility of PCR sequencing‐based technology.Methods Four HPV consensus primer sets（GP5＋/6＋,MGP,MY09/11,and PGMY09/11） were used in order to amplify a broad spectrum of HPV types for HPV infection in 325 cervical samples and the PCR products were sequenced afterwards for the HPV genotyping.Results The HPV‐positive rate was 75.4%,of which 35.5% harbored more than one HPV genotype.A total of 36 different genotypes was found,with HPV 16（24.1%） being the most prevalent,followed by HPV 58（13.3%） and HPV 52（9.6%）.There were substantial to almost perfect agreements between different primer sets regarding HPV detection efficiency,with the kappa value varying from 0.751 to 0.925,MGP,and PGMY09/11 were the most effective in detecting multiple infections（P0.001）.With each of the primer sets,a board range of HPV types could be identified,though there were several differences for a few genotypes.Conclusion The substantial agreement between PCR‐sequencing and HC2 for the detection of high‐risk HPV（kappa=0.761） indicated that PCR‐sequencing is also suitable for routine HPV screening.

A novel color image encryption scheme using fractional-order hyperchaotic system and DNA sequence operations

In this paper, Adomian decomposition method （ADM） with high accuracy and fast convergence is introduced to solve the fractional-order piecewise-linear （PWL） hyperchaotic system. Based on the obtained hyperchaotic sequences, a novel color image encryption algorithm is proposed by employing a hybrid model of bidirectional circular permutation and DNA masking. In this scheme, the pixel positions of image are scrambled by circular permutation, and the pixel values are substituted by DNA sequence operations. In the DNA sequence operations, addition and substraction operations are performed according to traditional addition and subtraction in the binary, and two rounds of addition rules are used to encrypt the pixel values. The simulation results and security analysis show that the hyperchaotic map is suitable for image encryption, and the proposed encryption algorithm has good encryption effect and strong key sensitivity. It can resist brute-force attack, statistical attack, differential attack, known-plaintext, and chosen-plaintext attacks.

Application of the first internal transcribed spacer(ITS-1)of ribosomal DNA as a molecular marker to population analysis in farrer's scallop Chlamys farreri

Sequence variation of the first internal transcribed spacer of ribosomal DNA （ ITS - 1 ） was examined and its application to the study of genetic variation was explored in four populations of farter＇ s scallop Chlamys farreri. ITS - 1 fragments, with a length of about 300 bp,of 78 individuals collected from Dalian, Qingdao, Yantai in China and Korea respectively were amplified via PCR, cloned and sequenced. Intra-genomic variation was examined by sequencing several clones of single individuals. Alignment and polymorphism analysis detected 44 haplotypes and 50 polymorphic sites which consist of 30 substitutions and 20 indels, indicating a high level of polymorphisms. Sequence analysis also showed a very low level of intra-individual variation. All these features validated the feasibility of application of ITS - 1 fragment to population analysis. Polymorphism analysis showed that the Korea sample has the richest genetic variation, followed by Yantai and Qingdao samples. AMOVA （analysis of molecular variance） showed that the majority （96.26%） of genetic variation was distributed within populations and 3.74% resulted from among populations, but with P 〈 0.05 （ = 0.042）, indicating that the populations in this study have significant divergence. This output was basically concordant with the result arising from RAPD data and different from that from mitochondrial 16S rDNA sequence data. Discussion on this inconsistency was made accordingly.

An Optimized Neural Network with Bat Algorithm for DNA Sequence Classification

Recently, many researchers have used nature inspired metaheuristicalgorithms due to their ability to perform optimally on complex problems. Tosolve problems in a simple way, in the recent era bat algorithm has becomefamous due to its high tendency towards convergence to the global optimummost of the time. But, still the standard bat with random walk has a problemof getting stuck in local minima. In order to solve this problem, this researchproposed bat algorithm with levy flight random walk. Then, the proposedBat with Levy flight algorithm is further hybridized with three differentvariants of ANN. The proposed BatLFBP is applied to the problem ofinsulin DNA sequence classification of healthy homosapien. For classificationperformance, the proposed models such as Bat levy flight Artificial NeuralNetwork (BatLFANN) and Bat levy Flight Back Propagation (BatLFBP) arecompared with the other state-of-the-art algorithms like Bat Artificial NeuralNetwork (BatANN), Bat back propagation (BatBP), Bat Gaussian distribution Artificial Neural Network (BatGDANN). And Bat Gaussian distributionback propagation (BatGDBP), in-terms of means squared error (MSE) andaccuracy. From the perspective of simulations results, it is show that theproposed BatLFANN achieved 99.88153% accuracy with MSE of 0.001185,and BatLFBP achieved 99.834185 accuracy with MSE of 0.001658 on WL5.While on WL10 the proposed BatLFANN achieved 99.89899% accuracy withMSE of 0.00101, and BatLFBP achieved 99.84473% accuracy with MSE of0.004553. Similarly, on WL15 the proposed BatLFANN achieved 99.82853%accuracy with MSE of 0.001715, and BatLFBP achieved 99.3262% accuracywith MSE of 0.006738 which achieve better accuracy as compared to the otherhybrid models.