Advances in microfluidic-based DNA methylation analysis

DNA methylation has been extensively investigated in recent years,not least because of its known relationship with various diseases.Progress in analytical methods can greatly increase the relevance of DNA methylation studies to both clinical medicine and scientific research.Microflu-idic chips are excellent carriers for molecular analysis,and their use can provide improvements from multiple aspects.On-chip molecular analysis has received extensive attention owing to its advantages of portability,high throughput,low cost,and high efficiency.In recent years,the use of novel microfluidic chips for DNA methylation analysis has been widely reported and has shown obvious superiority to conventional methods.In this review,wefirst focus on DNA methylation and its applications.Then,we discuss advanced microfluidic-based methods for DNA methylation analysis and describe the great progress that has been made in recent years.Finally,we summarize the advantages that microfluidic technology brings to DNA methylation analysis and describe several challenges and perspectives for on-chip DNA methylation analysis.This review should help researchers improve their understanding and make progress in developing microfluidic-based methods for DNA methylation analysis.

Analysis of DNA Cytosine Methylation on Cotton under Salt Stress

DNA methylation,especially methylation of cytosine in eukaryotic organisms,has been implicated in gene regulation,genomic imprinting,the timing of DNA replication,and determination of chromatin structure.It was reported that 6.5% of the whole cytosine residues in the nuclear DNA in

Analysis of heavy-ion-induced DNA strand breaks in plasmid pUC18

Plasmid DNA was irradiated or implanted by mixed particle field(CR) or lithium-ion-beam to detect strand breaks.The primary results showed that mixed particle field could induce single and double strand breaks with positive linear-dose-effects;most of sequence changes induced by CR were point mutant.Lithium-ion-beam could induce strand breaks also,but it was only at dose of 20Gy.

与黄原胶生物合成相关的“1.9”kb EcoRIDNA片段的序列测定分析

对Ｘａｎｔｈｏｍｏｎａｓｃａｍｐｅｓｔｒｉｓｐｖ．ｃａｍｐｅｓｔｒｉｓ（下称Ｘｃｃ）８００４菌株染色体基因组中９．４ｋｂＨｉｎｄⅢＤＮＡ的“１．９”ｋｂＥｃｏＲＩ酶切片段的测序分析结果表明，该ＥｃｏＲＩＤＮＡ片段的实际长度为１．８８ｋｂ。在核苷酸水平上与Ｘｃｃ的ｇｕｍ基因有９８％的一致性；这一ＥｃｏＲＩ片段上有两个有意义的ＯＲＦ：ＯＲＦ１和ＯＲＦ２。ＯＲＦ１是一个不完整的ＯＲＦ；在氨基酸水平上，ＯＲＦ１和ＯＲＦ２的推断性编码产物蛋白分别与ｇｕｍＡ基因编码的ＧｕｍＡ及ｇｕｍＢ基因编码的ＧｕｍＢ蛋白有１００％的一致性。因此在１．８８ｋｂＥｃｏＲＩ片段上存在一完整的ｇｕｍＢ基因。

Comparative analysis of dideoxy sequencing,the KRAS StripAssay and pyrosequencing for detection of KRAS mutation

AIM:To compare the differences between dideoxy sequencing/KRAS StripAssay/pyrosequencing for detection of KRAS mutation in Chinese colorectal cancer (CRC) patients.METHODS:Formalin-f ixed, paraff in-embedded (FFPE) samples with tumor cells ≥ 50% were collected from 100 Chinese CRC patients at Beijing Cancer Hospital. After the extraction of genome DNA from FFPE samples, fragments contained codons 12 and 13 of KRAS exon 2 were amplified by polymerase chain reaction and analyzed by dideoxy sequencing, the KRAS Strip Assay and pyrosequencing. In addition, the sensitivities of the 3 methods were compared on serial dilutions (contents of mutant DNA: 100%,50%,20%, 5%,10%, 5%,1%,0%) of A549 cell line DNA (carrying the codon 12 Gly>Ser mutation) into wild-type DNA (human normal intestinal mucosa). The results of dideoxy sequencing,the KRAS StripAssay and pyrosequencing were analyzed by Chromas Software, Collector forKRAS Strip Assay and the pyrosequencing PyroMarkTM Q24 system, respectively.RESULTS: Among 100 patients, KRAS mutations were identif ied in 34%, 37% and 37% of patients by dideoxy sequencing, the KRAS StripAssay and pyrosequencing, respectively. The sensitivity was highest with the KRAS Strip Assay (1%), followed by pyrosequencing (5%), and dideoxy sequencing was lowest (15%). Six different mutation types were found in this study with 3 main mutations Gly12 Asp (GGT>GAT), Gly12 Val (GGT>GTT) and Gly13 Asp (GGC>GAC). Thirty-three patients were identifi ed to have KRAS mutations by the 3 methods, and a total of 8 patients had conflicting results between 3 methods: 4 mutations not detected by dideoxy sequencing and the KRAS StripAssay were identified by pyrosequencing; 3 mutations not detected by dideoxy sequencing and pyrosequencing were identif ied by the KRAS StripAssay; and 1 mutation not detected by pyrosequencing was conf irmed by dideoxy sequencing and the KRAS StripAssay. Among these discordant results, the results identif ied by dideoxy sequencing were consistent either with the KRAS StripAssay or with pyrosequencing, which indicated that the accuracy of dideoxy sequencing was high. CONCLUSION: Taking a worldwide view of reports and our results,dideoxy sequencing remains the most popular method because of its low cost and high accuracy.

Relationship between HBV viremia level of pregnant women and intrauterine infection:neated PCR for detection of HBV DNA

ＲｅｌａｔｉｏｎｓｈｉｐｂｅｔｗｅｅｎＨＢＶｖｉｒｅｍｉａｌｅｖｅｌｏｆｐｒｅｇｎａｎｔｗｏｍｅｎａｎｄｉｎｔｒａｕｔｅｒｉｎｅｉｎｆｅｃｔｉｏｎ：ｎｅａｔｅｄＰＣＲｆｏｒｄｅｔｅｃｔｉｏｎｏｆＨＢＶＤＮＡＺＨＡＮＧＳｈｕＬｉｎ，ＨＡＮＸｉａｏＢ...

Nonextensivity and Tsallis Entropy in DNA Fragmentation Patterns by Ionizing Radiation

Nonextensive statistical mechanics as in Tsallis formalism was used in this study, along with the dynamical Hamiltonian rod-like DNA model and the maximum entropy criteria for Tsallis’ entropy, so as to obtain length distribution of plasmid fragments, after irradiation with very high doses, assuming that the system reaches metaequilibrium. By intensively working out the Grand Canonical Ensemble (used to take into account the variation of the number of base pairs) a simplified expression for Fragment Size Distribution Function (FSDF) was obtained. This expression is dependent on two parameters only, the Tsallis q value and the minimal length of the fragments. Results obtained from fittings to available experimental data were adequate and the characteristic behavior of the shortest fragments was clearly documented and reproduced by the model, a circumstance never verified from theoretical distributions. The results point to the existence of an entropy which characterizes fragmentation processes and depending only on the q entropic index.

Rapid detection of high-risk HPV16 and HPV18 based on microchip electrophoresis

Researches on detection of human papillomavirus(HPV)high-risk samples were carried out by polymerase chain reaction(PCR)coupled with microchip electrophoresis(MCE).Herein,we introduced a simple,rapid,automated method for detecting high-risk samples HPV16 and HPV18.In this research,general primers were initially selected to obtain sufficient detectable yield by PCR to verify feasibility of MCM method for HPV detection,then type-specific primers were further used to evaluate the specificity of MCE method.The results indicated MCE method was capable of specifically detecting high-risk HPV16 and HPV18,and also enabled simultaneous detection of multiplex samples.This MCE method described here has been successfully applied to HPV detection and displayed excellent reliability demonstrating by sequencing results.The inherent capability of MCE facilitated HPV detection conducted in a small chip with automated,high throughput,massive parallelized analysis.We envision that MCE method will definitely pave a way for clinical diagnosis,and even on-site screening of cervical cancer.

Molecular evolutionary analysis of gene families encoding DNA recombination and repair proteins and histone demethylases,and their functional implications

Many eukaryotic genes are members of multi-gene families due to gene duplications, which generate new copies that allow functional divergence. However, the relationship between

Molecular characterization and subtyping of Blastocystis in urticarial patients in Turkey

Objective:To investigate Blastocystis’etiologic role and association with gastrointestinal symptomatology in acute and chronic urticaria patients and to identify Blastocystis subtypes responsible for urticaria.Methods:The study included urticaria patients and healthy individuals that presented to our polyclinic between June 2015 and May 2017.The participants were assigned into Group栺(137 patients),subdivided into acute(72)and chronic urticaria patients(65),and Group栻(129 control individuals).Blastocystis presence was investigated by native-Lugol examination,trichrome staining,PCR using sequence tagged site primers,and DNA sequencing analysis.The phylogenetic tree was constructed.Results:The native-Lugol and trichrome staining methods revealed that 16 patients(16/133,12.0%)had Blastocystis-positive stool samples,of which seven samples(7/133,5.3%)belonged acute and nine(9/133,6.8%)to chronic urticaria patients.Concerning Blastocystis subtypes,of the acute urticaria patients,three had subtype 1(ST1),one had ST2,and three had ST3.Of the chronic urticaria patients,one had ST1 and eight had ST3.Blastocystis positivity was detected in two control individuals(2/123,1.6%),both being ST3.All subtypes identified by PCR were confirmed by the sequencing analysis.The acute and chronic urticaria groups showed no statistically significant differences for Blastocystis positivity(P=0.60)and subtype distribution(P=0.15).A statistically significant difference was found between the urticaria patients and the controls for Blastocystis positivity(P<0.01),but not for subtype distribution(P=0.67)or for Blastocystis presence and gastrointestinal complaints.Conclusions:This study on Blastocystis subtype distribution among Turkish urticaria patients showed results consistent with the literature.It was concluded that Blastocystis should be kept in mind in patients with urticaria.

An inter-laboratory study of DNA-based identity,parentage and species testing in animal forensic genetics

The probative value of animal forensic genetic evidence relies on laboratory accuracy and reliability.Inter-laboratory comparisons allow laboratories to evaluate their performance on specific tests and analyses and to continue to monitor their output.The International Society for Animal Genetics(ISAG)administered animal forensic comparison tests(AFCTs)in 2016 and 2018 to assess the limitations and capabilities of laboratories offering forensic identification,parentage and species determination services.The AFCTs revealed that analyses of low DNA template concentrations(≤300 pg/μL)constitute a significant challenge that has prevented many laboratories from reporting correct identification and parentage results.Moreover,a lack of familiarity with species testing protocols,interpretation guidelines and representative databases prevented over a quarter of the participating laboratories from submitting correct species determination results.Several laboratories showed improvement in their genotyping accuracy over time.However,the use of forensically validated standards,such as a standard forensic short tandem repeat(STR)kit,preferably with an allelic ladder,and stricter guidelines for STR typing,may have prevented some common issues from occurring,such as genotyping inaccuracies,missing data,elevated stutter products and loading errors.The AFCTs underscore the importance of conducting routine forensic comparison tests to allow laboratories to compare results from each other.Laboratories should keep improving their scientific and technical capabilities and continuously evaluate their personnel’s proficiency in critical techniques such as low copy number(LCN)analysis and species testing.Although this is the first time that the ISAG has conducted comparison tests for forensic testing,findings from these AFCTs may serve as the foundation for continuous improvements of the overall quality of animal forensic genetic testing.

Ecological distribution of harmful epiphytic Oscillatoriales in Alexandria coast,Egypt,with special reference to DNA identification

Objective:To identify the potentially harmful epiphytic Oscillatoriales species and follow up their distribution along Alexandria coast.Methods:Samples were collected bimonthly from April 2009 to February 2010 at three sites along Alexandria coast.Both morphological and molecular analyses were used for identifying the dominant species.Results:Five species belonging to two families were identified;Oscillatoria acutissima,Oscillatoria nigroviridis,Oscillatoria sp.,Lyngbya majuscule and Phormidium formosum.Their cell density ranged from 10^(3)to 126×10^(3)filament g^(-1)fresh weight macroalgae.The morphological study of the dominant species,Oscillatoria sp.(Oscillatoria sp.W1)showed much similarity with Planktothrix agardhii with no heterocysts and akinetes,while molecular ananlysis(16S rDNA)clustered the species in the same group with Anabaena sp.Conclusions:The 16S rDNA genes are not suitable for identifying Oscillatoriales during the present study and another molecular method should be used instead.

Evaluation of the AGCU Expressmarker 16 and 22 PCR Amplification Kits Using Biological Samples Applied to FTA Micro Cards in Reduced Volume Direct PCR Amplification Reactions

This study evaluated the performance of the Wuxi AGCU ScienTech Incorporation(HuiShan,Wuxi,China)AGCU Expressmarker 16(EX 16)and 22(EX22)short tandem repeat(STR)amplification kits in reduced reaction volumes using direct polymerase chain reaction(PCR)amplification workflows.The commercially available PowerPlex21(PP21)System(Promega,Wisconsin,USA),which follows similar direct workflows,was used as a reference.Anticoagulate blood applied to chemically impregnated FTATM Micro Cards(GE Healthcare UK Limited,Amersham Place,Little Chalfont,Buckinghamshire,HP79NA,UK)was used to represent a complex biological sample.Allelic concordance,first‑pass success rate,average peak heights,heterozygous peak height ratios(HPHRs),and intracolor and intercolor peak height balance were determined.In reduced volume PCR reactions,the performances of both the EX16 and EX22 STR amplification kits were comparable to that of the PP21 System.The level of performance was maintained at PCR reaction volumes,which are 40%of that recommended.The EX22 and PP21 System kits possess comparable overlapping genome coverage.This study evaluated the performance of the AGCU EX16 and EX22 STR amplification kits in reduced PCR reaction volumes using direct workflows in combination with whole blood applied to FTATM Micro Cards.Allelic concordance,first‑pass success rate,average peak heights,HPHRs,and intracolor and intercolor peak height balance were determined.A concordance analysis was completed that compared the performance of the EX16 and EX22 kits using human blood applied to FTA Micro Cards in combination with full,half,and reduced PCR reaction volumes.The PP21 System(Promega)was used as a reference kit.Where appropriate,the distributions of data were assessed using the Shapiro‑Wilk test.For normally‑distributed data,statistics were calculated using analysis of variance(ANOVA)and for nonparametric data the Wilcoxon/Kruskal‑Wallis test was used.Statistical significance was set at P<0.05.Confidence intervals for mean values were set at 95%.On using reduced volume PCR reactions in combination with dried blood spots applied to FTA sample collection cards,both the EX16 and EX22 kits were shown to generate STR profiles of sufficient quality to allow entry into National DNA databases.The performance of both EX16 and EX22 was comparable to that of the PP21 System.This study demonstrates the successful use of the Wuxi AGCU ScienTech Incorporation EX16 and EX22 kits in reduced PCR reaction volumes with complex biological samples applied to chemically impregnated FTA sample collection cards.

Distribution and space use of seed-dispersing rodents in central Pyrenees:implications for genetic diversity,conservation and plant recruitment

The function and conservation of many forest ecosystems depend on the distribution and diversity of the community of rodents that consume and disperse seeds.The habitat preferences and interactions are especially relevant in alpine systems where such granivorous rodents reach the southernmost limit of their distribution and are especially sensitive to global warming.We analyzed the community of granivorous rodents in the Pyrenees,one of the southernmost mountain ranges of Europe.Rodent species were identified by DNA with particular attention to the Apodemus species,which are prominent seed-dispersing rodents in Europe.We confirmed for the first time the presence of the yellow-necked mouse,Apodemus flavicollis,in central Pyrenees,a typical Eurosiberian species that reaches its southernmost distribution limit in this area.We also found the wood mouse,Apodemus sylvaticus,a related species more tolerant to Mediterranean environments.Both rodents were spatially segregated by altitude.A.sylvaticus was rare at high altitudes,which might cause the genetic differentiation between populations of the different valleys reported here.We also found other seed consumers like dormice,Elyomis quercinus,and voles,Myodes glareolus,with marked habitat preferences.We suggest that population isolation among valleys may increase the genetic diversity of rodents,like A.sylvaticus.We also highlight the potential threat that global warming may represent for species linked to high-altitude refuges at the southern edge of its distribution,like Apodemus flavicollis.Finally,we discuss how this threat may have a dimension in the conservation of alpine forests dispersed by these rodent populations.

Y‑STR Kits and Y‑STR Diversity in the South African Population: A Review

The South African population consists of four ethnic groups,i.e.,Blacks,Coloreds,Indians,and Whites,and is considered the most diverse conglomeration of humans.In addition to autosomal short tandem repeat(STR)variation,an important tool to study population diversity is Y-chromosome(Y)-STR analysis.Y-STRs aid in forensic investigations and provide essential data about paternal lineage origins.Y-STR kits consisting of an array of stable and rapidly mutating markers offer crucial information on a given population’s genetic and haplotype diversity.This review discusses the development of Y-STR kits over the years and highlights some prominent Y-STR studies conducted on the South African population.The earliest Y-STR kit developed was the Y-PLEX™6,with the most recent being the UniQTyper™Y-10 Multiplex.The South African population studies show varying data,with the“minimal haplotype”having low discrimination capacity among the ethnic groups and the UniQTyper™Y-10 showing high genetic diversity among the ethnic groups of the country.There is a dearth of Y-STR studies on the South African population.With the advent of new Y-STR kits with increased discriminatory markers,additional studies are required to represent the South African population in the Y-STR databases.Considering the diversity of the South African population,establishment of a local/regional population database would be beneficial.In addition,data on the origins and prevalence of mutations and silent alleles should be obtained from STR datasets generated during kinship investigations(specifically,parentage tests)so that detailed information about the frequencies of mutations,silent alleles,and uniparental disomy in the South African population at Y STR loci can be estimated.

Identification of Colored Dyes that are Resistant to Fading on Exposure to Ethylene Oxide;Use with Indicating FTA™Sample Collection Cards

Regulatory Standards and Forensic Communities are expressing an expectation for HID products to be certified as“DNA‑free.”Recently,“DNA‑free”status was described for HID‑related products using ethylene oxide(EtO);this gas reduces the presence of amplifiable DNA and causes minimal interference to downstream HID‑analytical methods.During sample collection,indicating cards,for example,Indicating FTA™(GE Healthcare Life Sciences,UK),are used to collect and store buccal cell DNA.These cards contain a dye which changes color on application of a colorless sample.Generating“DNA‑free”indicating cards using EtO should not impact the dyes’ability to indicate sample location or the efficacy of the card in downstream HID‑analytical methods.This study was initiated to identify alternative dyes to those currently used with sample indicating collection cards.The most promising,dyes when applied to cellulose papers exhibited a uniform color distribution and excellent sample indicating properties even when mixed with chemicals associated with FTA™.When dyed cellulose papers were exposed to EtO,ultraviolet radiation,elevated temperature,and humidity,negligible fading or discoloration was observed.The presence of these dyes on cellulose papers did not interfere with direct short tandem repeat(STR)profiling.Allelic concordance,first pass success rate,and mean peak heights were comparable to samples applied to Indicating FTA.Biological samples applied to EtO‑treated dyed cellulose papers and stored>1 month produced full STR profiles of sufficient quality to allow submission to DNA databases,confirming negligible interference from EtO treatment.These alternative sample indicating dyes resist EtO‑mediated fading while fulfilling the Forensic Community’s expectation for“DNA‑free”with negligible impact on collection card performance.

Fungal diversity in adult date palm(Phoenix dactylifera L.) revealed by culture-dependent and culture-independent approaches

Endophytic flora plays a vital role in the colonization and survival of host plants, especially in harsh environments, such as arid regions. This flora may, however, contain pathogenic species responsible for various troublesome host diseases. The present study is aimed at investigating the diversity of both cultivable and non-cultivable endophytic fungal floras in the internal tissues(roots and leaves) of Tunisian date palm trees(Phoenix dactylifera). Accordingly, 13 isolates from both root and leaf samples, exhibiting distinct colony morphology, were selected from potato dextrose agar(PDA) medium and identified by a sequence match search wherein their 18S–28S internal transcribed spacer(ITS) sequences were compared to those available in public databases. These findings revealed that the cultivable root and leaf isolates fell into two groups, namely Nectriaceae and Pleosporaceae. Additionally, total DNA from palm roots and leaves was further extracted and ITS fragments were amplified. Restriction fragment length polymorphism(RFLP) analysis of the ITS from 200 fungal clones(leaves: 100; roots: 100) using HaeIII restriction enzyme revealed 13 distinct patterns that were further sequenced and led to the identification of Alternaria, Cladosporium, Davidiella(Cladosporium teleomorph), Pythium, Curvularia, and uncharacterized fungal endophytes. Both approaches confirmed that while the roots were predominantly colonized by Fusaria(members of the Nectriaceae family), the leaves were essentially colonized by Alternaria(members of the Pleosporaceae family). Overall, the findings of the present study constitute, to the authors' knowledge, the first extensive report on the diversity of endophytic fungal flora associated with date palm trees(P. dactylifera).