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.

Sequence Analysis of Mitochondrial DNA D-loop Region in Xinjiang Goose

[Objective] The sequences of mitochondrial DNA D-loop region of Xinjiang Goose with three different colors of plumage were analyzed in order to study the genetic diversity of Xinjiang Goose, as well as the phylogeny and evolution. [Method] Ten geese were selected randomly from the core populations of grey-, mosaic- and white-plumaged Xinjiang Goose respectively with a total number of thirty as experi- mental materials, of which the blood samples were collected from the largest vein under the wing （brachial vein） for DNA extraction. Sequences of mitochondrial DNA D-loop regions were determined using DNA sequencing technology to analyze the polymorphism. In addition, the genetic distances among different populations were estimated through the comparison with the reference sequences. [Resull] The con- tents of A, G, C and T nucleotides in the D-loop region of Xinjiang Goose were 28.85%, 17.05%, 25.38% and 28.72%, respectively. The average haplotype diversity and nucleotide diversity of Xinjiang Goose were 0.583 and 0.056. Xinjiang Goose and Greylag Goose were clustered into the same group. [Conclusion] The results showed that Xinjiang Geese with three different colors of plumage all descend from Greylag Goose （Anser anser）.

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

Improvements on environmental DNA extraction and purification procedures for matagenomic analysis

Our previously described environmental DNA extraction method has been widely used in environmental microbial community analysis. However, residual humic substances may remain with obtained environmental DNA, which interferes downstream molecular analyses. To remedy this situation, two DNA extraction buffers （PIPES and Tris-HCl） and four purification strategies including our new modified low melting point gel purification method and three commercial kits from QIAEX, Omega and Promega were evaluated with diverse soil samples. The PIPES buffer （pH 6.5） is found to be more effective for removing the humic substances, but it leads to lower DNA yield and causes more severe DNA shearing than using the Tris-HC1 buffer （pH 8.0）. Gel purification and the Promega purification kit achieve much higher DNA recoveries than QIAEX or Omega kit, and higher purity of DNA is obtained by gel purification than by the Promega kit with both DNA extraction buffers mentioned above. Considering all results together, two alternative methods for DNA extraction and purification are proposed： one uses Tris-HCl buffer extraction and gel purification as the primary approach when the amount of soil or biomass is not a major concern, and the other uses PIPES buffer extraction and the Promega kit purification when severe DNA shearing and/or limited biomass occurs. Purified DNA samples by both methods are amenable for use as templates for whole community genome amplifications and PCR amplifications of bacterial 16S rRNA genes. It is demonstrated that these two alternative methods could be applied to a wide variety of environmental samples.

Aptamer-Based DNA Materials for the Separation and Analysis of Biological Particles

DNA is a biological macromolecule that carries genetic information in organisms.It provides a series of predominant bio-logical advantages,such as sequence programmability,high biocompatibility,and low biotoxicity.As such,it is a unique polymer material that shows great potential for application in biological and medical fields.DNA aptamers are short DNA sequences with a specific ability of molecular recognition.With its discovery,the application prospect of DNA materials has broadened,especially for the separation and analysis of biological particles.In this review,the functions and characteristics of DNA aptamers are introduced,and the applications of DNA materials in cell/exosome separation and cancer detection are summarized.The application prospect and possible challenges of DNA materials are predicted.

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.

FLOW CYTOM ETRIC ANALYSIS OF CELLULAR DNA CONTENT IN EPITHELIAL OVARIAN TUMOR

The DNA content of tumor all was analyzed by flow cytometry on parafflnembedded specimens in 73 patients with epithelial ovarian tumor, and its clinical significance was evaluated. One of the 5 benign (20%), 2 of the 11 borderline (18.18%), and 30 of the 57 malignant (52. 63%) tumors were aneuplold. The occurrence rate of aneuploidy In malignant tumors was higher than In benign and borderline tumors ( P < 0. 05 ). Furthermore, aneuploidy was more frequently In the advanced stages (Ⅲ -Ⅳ ) (77. 7%) than in the early stages (Ⅰ - Ⅱ ) (9. 5%) (P<0. 005). The occurrence rate of DNA aneuploidy was higher in patients associated with ascites and the residual tumor≥.2 cm. Patients with aneuploid tumors had more of ten ascites (P<0. 005) and residual tumor size≥2cm (P< 0.005). There was no apparent correlation between the DNA ptoidy and the histologic grade, histologic type of the tumors. G0/G1 cell proportion of DNA diplold tumors in advanced carcinoma (64. 6%) was less than those of early stage carcinoma (75. 9% ) (P<0. 05). The survival rate of diplold tumor patients was higher than that of aneuploid tumor patients in the different time after operation, and the median survival time was 30. 2 months and 10. 3 months, respectively. Multivariate analysis revealed that cellular DNA ploidy was the most Important predictive factor (P = 0. 007) of prognosis, followed by residual tumor size (P= 0. 05). Different tumor specimen of the same patient can exhibit variation sometime (38. 9%).The results revealed that the DNA ploidy may reflect tumor biological characteristics, I. e. , Its proliferative ability. Analysis of cellular DNA content of epithelial ovarian tumors would help us to predict the prognosis of the patients better.

Iron-Mediated Oxidative DNA Damage Detected by Fluorometric Analysis of DNA Unwinding in Isolated Rat Liver Nuclei

Studies were performed to determine the extent of nuclear DNA degradation induced by iron, iron-ascorbate, or iron-bleomycin under aerobic conditions in a model system using isolated rat liver nuclei. The effects of five antioxidants (catalase, superoxide dismutase, dimethyl sulfoxide, glutathione and diallyl sulfide) on this oxidative nuclear damage were also investigated. At the 0.05 level for statistical significance, iron induced concentration-dependent DNA degradation, and this effect was enhanced by ascorbate and bleomycin. The antioxidants catalase, dimethyl sulfoxide, and diallyl sulfide significantly reduced the iron-ascorbate-induced DNA damage, whereas superoxide dismutase and dimethyl sulfoxide significantly reduced iron-bleomycin-induced damage. Glutathione significantly increased the iron-bleomycin-induced DNA damage. These results suggest that the reactive oxygen species generated by iron, iron-ascorbate, and iron-bleomycin are responsible for the DNA strand breaks in isolated rat liver nuclei.

Correlative Analysis on the Relationship between PMI and DNA Degradation of Cell Nucleus in Human Different Tissues

Summary： To determining the postmortem interval （PMI） through quantitative analysis of the DNA degradation of cell nucleus in human brain and spleen by using image analysis technique （IAT）. The brain and spleen tissues from 32 cadavers with known PMI were collected, subjected to cell smear every 1 h within the first 5-36 h after death, stained by Feulgen-Van＇s staining, Three indices reflecting DNA in brain cells （astrocytes） and splenic lymphocytes, including integral optical density （IOD）, average optical density （AOD）, average gray （AG） were measured by employing the mage analysis instrument. The results showed that IOD and AOD declined and AG increased with the prolongation of dead time within 5 36 h. A correlation between the PMI and gray parameters （IOD,AOD and AG） was identified and the corresponding regression equation was obtained. The parameters （IOD, AOD and AG） were proved to be effective quantitative indicators for accurate estimation of PMI within 5-36 h after death.

Analysis of Mitochondrial DNA from the Ancient Tombs of Turfan

MtDNA was successfully extracted from ten individual bones (femurs) in the tombs of ancient Jushi in Turfan basin, dated back to the year about 3 000-2 500 years ago. By means of four overlapping primers, we got nucleotide sequence of the 218bp length. Ancient mtDNA was analyzed by the sequencing of hypervariable region Ⅰ of the mtDNA control region. The result shows that 9 haplotypes with 24 polymorphic sites were obtained. The phylogenetic analysis indicated that Mongolians and Altai are the population genetically closest to the Jushi groups and Jushi mtDNA pool being an admixture of eastern Asian and European lineages. So our preliminary data imply that an ancient mingling of Euro-Asian population had existed in Turfan basin prior to the early Iron Age.

Image Analysis for Degradation of DNA in Retinal Nuclei of Rat after Death

The changes of retinal nuclear DNA content in rats after death was detected and the relationship between degradation of retinal nuclear DNA and postmortem interval （PMI） was analyzed. Ninety healthy adult SD rats, female, weighing 250±10 g, were randomly divided into 15 groups. At 20 ℃, the retinal cells were withdrawn every 2 h within 0 to 28 h after death and stained with Feulgen-Vans. Index of density （ID）, integral absorbance （IA） and average absorbance （AA） in retinal nucleus were analyzed by image analysis system. And the obtained data were subjected to linear regression analysis by using SPSS12.0 software. The results showed that in retinal nucleus, AA and IA were gradually declined with the prolongation of PMI, while ID had an increased tendency. Within 28 h after PMI, the regression equations were as follows： YAA=-0.009XAA＋0.590 （R^2=0.949）, YIA=0.097XIA＋18.903 （R^2=0.968）, YID=0.122XID＋2.246 （R^2=0.951）. It was concluded that retinal nuclear DNA after death in rats was degraded gradually and had a good correlation with PMI.

The Biodiversity of Shrimp Genus Artemia from Russian Lakes:Morphometric,Cytogenetics and DNA-analysis

Shrimps of genus Artemia are the inhabitants of continental and marine waters with salinity of 70 to 350 g/l and above.Artemia is able to survive in the conditions in which other animals cannot exist.This is due to adaptations:effective osmoregulation system,the ability to synthesize of respiratory pigment(hemoglobin)and diapauses cysts(Litvinenko at.al.,2009).Cysts of this

Normal Mode Analysis on Three Different Structures of a Duplex DNA d(CGCGAATTCGCG)

Normal mode analysis in dihedral angle space was carried out on two X ray crystal structures and one model structure responded to the same sequence of duplex DNA: d(CGCGAATTCGCG). Comparing these results indicates that it is reliable and meaningful to carry out normal mode analysis on model structures. The reliability is greater except for the ends of helix.

Determination of the Early Time of Death by Computerized Image Analysis of DNA Degradation: Which Is the Best Quantitative Indicator of DNA Degradation?

This study evaluated the correlation between DNA degradation of the splenic lymphocytes and the early time of death, examined the early time of death by computerized image analysis technique （CIAT） and identified the best parameter that quantitatively reflects the DNA degradation. The spleen tissues from 34 SD rats were collected, subjected to cell smearing every 2 h within the first 36 h after death, stained by Feulgen-Van＇s staining, three indices reflecting DNA content in splenic lymphocytes, including integral optical density （IOD）, average optical density （AOD）, average gray scale （AG） were measured by the image analysis. Our results showed that IOD and AOD decreased and AG increased over time within the first 36 h. A stepwise linear regression analysis showed that only AG was fitted. A correlation between the postmortem interval （PMI） and AG was identified and the corresponding regression equation was obtained. Our study suggests that CIAT is a useful and promising tool for the estimation of early PMI with good objectivity and reproducibility, and AG is a more effective and better quantitative indicator for the estimation of PMI within the first 36 h after death in rats.

Mitochondrial DNA Markers for PCR-Based Phylogenetic Analysis of Ark Shells

Arcidae species are commercially important bivalves in Japan and are commonly referred to as bloody ark due to their red blood. They have thick shells with distinct radiating ribs, and the numbers of these ribs are important morphological features for species discrimination. However, some Arcidae species are morphologically indistinguishable, with a similar number of the ribs in adults and deficient rib formation, particularly among juveniles. Thus, we developed a reliable molecular marker to genetically discriminate between 7 Arcidae species belonging to Scapharca, Anadara, and Tegillarca based on species-specific polymorphic segments of mitochondrial DNA. PCR amplification of partial COI, 16S rRNA, 12S rRNA, and Cyt b genes was performed on 7 species using 8 primer sets. Only the set of Scapharca-specific forward primer and universal reverse primer for the partial COI gene successfully yielded single PCR products from all 7 species examined. Thus, nucleotide sequences of 481 bp portion of these PCR products were determined, and the degrees of nucleotide substitutions ranged from 0.4% between S. broughtonii and T. granosa to 20.2% between S. satowi and A. antiquata. In addition, a phylogenetic tree showed significant differences between 7 species, with higher bootstrap support than 69.

Study on the Detection of Telomerase Activity by Combining DNA Sequence Analysis with TRAP

Telomeric repeat amplification protocol (TRAP) is now a conventional assay for detecting telomerase activity. However, this method presents problems owing to tedious quantitation, radioisotopic handling. In order to alleviate these inconveniences, a novel telomerase DNA sequencing assay together with TRAP to detect human telomerase activity was developed. It was used to detect telomerase activity in Hela, HLF, MCF, K562, SMMC 7721 cells, Leukocytes and RNase pretreated or heat treated cells as control. Telomerase activity assayed by this method was positive when the number of K562 cells examined was 102,103, and 104. The telomerase activity depended on the number of K562 cells used in the assay. Telomerase activity of Rnase pretreated cells or heat treated cells, and human normal peripheral blood leukocyte(Leu) were negative. The result of this method was available within a few hours and was handled without radioisotope. Further studies should be taken to detect telomerase activity in quantitation.

Utilizing Short Tandem Repeats (STRs) as a Resolving Matrix in Parental Dispute DNA Analysis

Interest in DNA analysis using short tandem repeats (STR) as finger printing tools in forensic medicine has gained tremendous application, as expression of these nuclear factors have enhanced forensic examination. Here we used this Biochemical characterization after conventional extraction process, polymerase chain reaction (PCR), gel electrophoresiss and a sequencer to distinguish and resolve parental dispute. The differential migration of labeled DNA fragments which attains excitation energy with a laser elicits fluorescent light of different wavelength depending on the dye used. A data collection software (Genemapper) collects raw data (spectrograph) and converts it to an electropherogram that is interpreted. By comparing the DNA profiles, inclusion and exclusion criteria were elucidated to resolve disputes. The inherent discriminating power of STRs used in analysis enhances resolution of cell mixtures, genetic aberration, substantiation of tissue origin and provides genetic distinction which is a robust and reliable approach in resolving parental disputes.

A New Numerical Method for DNA Sequence Analysis Based on 8-Dimensional Vector Representation

Background: The multiple sequence alignment (MSA) algorithms are the traditional ways to compare and analyze DNA sequences. However, for large DNA sequences, these algorithms require a long time computationally. Objective: Here we will propose a new numerical method to characterize and compare DNA sequences quickly. Method: Based on a new 2-dimensional (2D) graphical representation of DNA sequences, we can obtain an 8-dimensional vector using two basic concepts of probability, the mean and the variance. Results: We perform similarity/dissimilarity analyses among two real DNA data sets, the coding sequences of the first exon of beta-globin gene of 11 species and 31 mammalian mitochondrial genomes, respectively. Conclusion: Our results are in agreement with the existing analyses in our literatures. We also compare our approach with other methods and find that ours is more effective.

DNA extraction from archived hematoxylin and eosin-stained tissue slides for downstream molecular analysis

BACKGROUND Histopathologically stained archived tissue slides are stored in hospital archives for years to decades.They are the largest available source of biological materials and are a potentially useful resource that can be used for retrospective epidemiological studies.DNA recovered from the slides can be used for several downstream molecular processes including polymerase chain reaction,single nucleotide polymorphism analysis,and whole genome sequencing.The DNA from these slides can be utilized to compare gene signatures of normal and diseased tissues.However,extraction of high-quality DNA from archived stained hematoxylin and eosin(H&E)slides remains challenging.AIM To standardize a new protocol for extracting DNA from archived H&E-stained tissue slides for further molecular assays.METHODS A total of 100 archived H&E-stained cancer slides were subjected to a total of five methods of DNA extraction.Methods were varied in the deparaffinization step,tissue rehydration,duration of lysis,and presence or absence of proteinase K.The extracted DNA was quantified using a NanoDrop spectrophometer and the quality was analyzed by agarose gel electrophoresis.Then each sample was subjected to polymerase chain reaction(PCR)to amplify the internal control gene GAPDH,thereby confirming the DNA intactness,which could be further utilized for other downstream applications.RESULTS Of the five different methods tested,the third method wherein xylene was used for tissue deparaffinization followed by 72 h of digestion and without proteinase K inactivation yielded the highest amount of DNA with good purity.The yield was significantly higher when compared to other methods.In addition,90%of the extracted DNA showed amplifiable GAPDH gene.CONCLUSION Here we present a step-by-step,cost-effective,and reproducible protocol for the extraction of PCR-friendly DNA from archived H&E-stained cancer tissue slides that can be used for further downstream molecular applications.

Analysis on the development status of DNA storage based on papers and patents

DNA storage as a disruptive technology is expected to solve the problem of massive data storage.Based on bibliometric analysis of DNA storage related papers and patents,this paper analyzes the development trend of DNA storage technology.The results show that DNA technology is still in the development stage,in which only a small number of researchers are involved.USA is the global leading country of DNA storage research.Both universities and companies in USA have played an important role in promoting DNA storage research.China is second only to USA in the number of DNA storage related papers or patents.However,in terms of patents layout,Chinese institutions don’t have sufficient intention of opening up the global market in the application of DNA storage.Although there have been several breakthrough advances in DNA storage,there are still many challenges to be solved.