A Method Suitable for Extracting Genomic DNA from Animal and Plant——Modified CTAB Method

[Objective] The study aimed to introduce a rapid and effective method that is suitable for extracting genomic DNA from animal and plant. [ Method ] The genomic DNAs were extracted from tender leaves of 24 peanut cuhivars and from the liver, lung and kidney of white mouse through the specifically modified CTAB method. The DNAs were run on agarose gel, next detected by DNA/Protein analyzer. Finally PCR amplification was conducted to detect the quality of DNAs extracted using the modified CTAB method. [ Result] The clear and orderly bands were observed in gel detection, and the values of OD200/OD200 for DNAs extracted via modified CTAB method were between 1.77 - 1.83. The DNAs performed well in PCR amplification. [ Conclusion] The DNAs extracted by modified CTAB method could satisfy the requirement of PCR amplification.

Comparative Study on Four Methods for Quick Extraction of Sorghum Genomic DNA

[Objective] This study was to find out a quick,simple,and low-cost method for the extraction of sorghum genomic DNA.[Method] Four plant genomic DNA extraction methods based on CTAB,including liquid nitrogen grinding method（method I）,buffer grinding method（method II）,drying grinding method（method III）and directly grinding method（method IV）,were used to extract the sorghum genomic DNA from leaves;further the quantity and quality of the yielded DNA were detected by gel electrophoresis,SSR-PCR and SRAP-PCR.[Result] These four methods performed no remarkable difference in DNA product.The method I and method II produced DNA with higher purity and better integrity,which,especially from method I,is effective for SRAP-PCR and SSR-PCR.While the DNA extracted via method III and method IV had less integrality and lower purity,and only effective in SSR-PCR.[Conclusion] Enough amount of sorghum genomic DNA to perform tens of PCR could be quickly extracted using all these four methods.The DNA obtained via method I and method II had a broader application spectrum（SRAP,RAPD,ISSR and SSR）than that via method III and method IV which is only proper for PCR targeting small DNA fragments（SSR）.

Comparison on Four Extraction Methods of Genomic DNA from Clematis fasciculiflora Franch

[Objective] This study aimed at comparing the four extraction methods of genomic DNA from Clematis fasciculiflora Franch and determining the optimal extraction method for extracting the genomic DNA from Clematis fasciculiflora Franch.[Method] Leavies of Clematis fasciculiflora Franch were used as materials for comparing the purity and concentration of extracted DNA and extracting time among the four extraction methods of genomic DNA including improved CTAB method Ⅰ,improved CTAB method Ⅱ,improved CTAB method Ⅲ and improved SDS method.[Result] The four extraction methods could all be successfully used for extracting the genomic DNA from Clematis fasciculiflora Franch.The purity of genomic DNA was the highest using improved CTAB method Ⅰ,with the longest extracting time;while the concentration of genomic DNA was the maximum using the improved SDS method,with the shortest extracting time and relatively low purity;the extracting time of improved CTAB method Ⅲ was the shortest.[Conclusion] This study had established the optimal extraction method for extracting the genomic DNA from Clematis fasciculiflora Franch and supported for the further research using molecular biological methods.

A Method for Rapid Salt-extraction of High-quality Genomic DNA from Plant Seeds

[Objective] This study aimed to explore an effective method for rapid salt- extraction of high-quality genomic DNA from dried seeds of plants. [Method] Seeds of seven varieties of crops were ground into powder. A hundred milligrams of seed powder was added to extracting solution for high salt-extraction of genomic DNA. The yield and quality of extracted DNA were determined by using ultramicro UV/Vis spectrophotometer detection method, PCR and restriction enzyme digestion. [Result] About 619.67-1 811.21 ng of genomic DNA was extracted from per 100 mg of dried seed powder of seven varieties of conventional crops. A260/A280 ratios of the obtained DNA solution all ranged from 1.87 to 2.07, the purity and quality of PCR were suitable for PCR and restriction enzyme digestion. Clear target bands of specific endogenous gene fragments of seven varieties of crops were amplified by PCR, and the obtained DNA could be fully digested with EcoRV and Hindlll.[Conclusion] This method could be used for rapid extraction of high-quality genomic DNA from dried seeds.

A New Sampling Approach to Obtain Genomic DNA of Marine Shellfish

[Objective] The aim was to explore a new sampling approach to obtain genomic DNA of marine shellfish,as well as to provide reference for the molecular biology research on precious shellfish.[Method] Meretrix meretrix,Atrina pectinata,Perna viridis,Crassostrea hongkongensis and Scapharca kagoshimensis were used as experimental materials and the genomic DNA of adductor muscle was taken as reference to extract the genomic DNA of shell cavity fluids with the conventional phenol-chloroform method.And then biophotometer,agarose gel electrophoresis,amplification and sequencing of the target fragments were used to examine the quality of genomic DNA.At the same time,the phylogenic tree was constructed to verify the reality of source contributions.[Result] The quality of genomic DNA of shell cavity fluids extracted by the phenol-chloroform method was better than the genomic of adductor muscle.The genomic DNA extracted by this method showed less content of protein,polyphenol and pigment,which could completely meet the demands of amplification and sequencing of the target fragments.Through the phylogenic tree,it was verified that the source contributions of shell cavity fluids were not come from foreign pollutions.[Conclusion] It is completely feasible to obtain the genomic DNA from shell cavity fluids,which could be applied in the target fragments amplification.

A Rapid and Efficient Method for Preparing Genomic DNA from Microbacterium sp.

A new method was used to preparing genomic DNA from Microbacterium sp.quickly and efficiently.DNA quantity and purity was measured by UV absorbance.Integrity of the genomic DNA was tested by agarose gel eletrophoresis.The DNA prepared by this method was sufficiently pure for PCR.This method saves time and cost,practices easily as well.

Comparison of Different Extraction Methods of Genomic DNA of Raw Soybean Milk

[Objective] The paper was too explore and compare methods of DNA extraction from raw soybean milk.[Method] Taken the soybean milk purchased from market as the material,pyrolysis method,isopropanol precipitation method,CTAB method,SDS method,high-salt low-pH and guanidine isothiocyanate method,as well as their improved methods were used to extract genomic DNA,and the extraction effects of these methods were compared by detecting the DNA using optical density,agarosegel electrophoresis and polymerase chain reaction（PCR）methods.[Result] The genomic DNA extracted by all methods except isopropanol precipitation method could be used in PCR reaction.Meanwhile,the high DNA concentration and purity will be gained by different methods in the order of high-salt low-pH method,high-salt low-pH method,improved CTAB method,improved isopropanol precipitation method,guanidine isothiocyanate method and improved pyrolysis method.[Conclusion] These methods are simply to operate,fast to gain results,and suitable for the extraction of total DNA from raw soybean milk.

Genetic Analysis of Genomic DNA in Shanxi Lean Meat Pigs (SD-Ⅲ Line)

[Objective] This study aimed to detect the purity of Shanxi lean meat pigs. [Method] Eight primers were selected and used in the DNA fingerprinting technique amplified fragment length polymorphism (AFLP) to detect the purity of 30 Shanxi lean meat pigs. [Result] A total of 206 AFLP markers were obtained; 3-15 bands were obtained from each primer. The similarity index of population in Shanxi local pigs was 0.931 on average. [Conclusion] AFLP is suitable for detection of genomic DNA, and the genetic purity of SD-Ⅲ line is higher.

A simple method for purification of genomic DNA from whole blood using Fe_3O_4/Au composite particles as a carrier

Objective： To establish a method of genomic DNA extraction from whole blood using Fe3O4/Au composite particles as a carrier. Methods： Two crucial conditions （sodium chloride concentration and amount of the magnetic particles） were optimized and 8 different human whole blood samples were used to purify genomic DNA under the optimal condition. Then agarose gel electrophoresis and polymerase cbain reaction （PCR） were performed. Results： The optimal binding condition was 1.5 mol/L NaC1/10% PEG, and the optimal amount of Fe3O4/Au composite particles was 600μg. The yields of the genomic DNA from 100μl of different whole blood samples were 2-5 μg, and the ratio of A260/A280 was in the range of 1.70-1.90. The size of genomic DNA was about 23 kb and the PCR was valid. Conclusion： The purification system using Fe3O4/Au composite microparticles has advantages in high yield, high purity, ease of operating, time saving and avoiding centrifugation. The purified sample was found to function satisfactorily in PCR amplification.

Preparation of high molecular weight （HMW） genomic DNA from tea plant （Camellia sinensis） for BAC library construction

A bacterial artificial chromosome （BAC） library is an invaluable resource tool to initiate tea plant genomics research, and the preparation of high molecular weight （HMW） genomic DNA is a crucial first step for constructing a BAC Library. In order to construct a BAC library for enhancing tea plant genomics research, a new method for the preparation of tea pant high molecular weight （HMW） genomic DNA must be developed due to young tea plant leaves and shoots are notably rich in both tea polyphenols and tea polysaccharides. In this paper, a modified method for preparing high quality tea plant HMW genomi~ DNA was optimized, and the quality of tea plant genomic DNA was evaluated. The results were as follows： Critical indicators of HMW DNA preparation were the appearance of the smooth nuclei in solution （as opposed to sticky-gummy） before agarose plug solidification, non-dark colored nuclei plugs after lysis with an SDS/proteinase K solution, and the quality and quantity of HMW DNA fragments after restriction enzyme digestion. Importantly, 1% dissolved PVP-40 and 1% un-dissolved PVP-40 during the nuclei extraction steps, in conjunction with the removal of PVP-40 from the plug washing and nuclei lysis steps, were critical for achieving HWM tea plant DNA suitable for BAC library construction. Additionally, a third PFGE fraction selection step to eliminate contaminating small DNA fragments. The modifications provided parameters that may have prevented deleterious interactions from tea polyphenols and tea polysaccharides. The HMW genomic DNA produced by this new modified method has been used to successfully construct a large-insert tea plant BAC library, and thus may be suitable for BAC library construction from other plant species that contain similarly interfering compounds.

Affinity chromatography-dependent selection (ACDS)of genomic DNA fragments bound specifically to bacterial synthesized Myc/Myn proteins

This paper describes an approach to seek for mouse c-Myc/Myn proteins-bound specific sequences among ge-nomic DNA. cDNA fragment of myn gene was obtained through RT-PCR technique from RNA of NIH3T3 cells. DNA fragments encoding BR/HLH/LZ structure of Myc and Myn proteins were cloned in frame into pGEX-2T vec-tor respectively Fusion GST-Myc and GST-Myn synthe-sized in E.coli hosts showed affinity to CACGTG E-boxDNA and subsequently interacted with genomic fragments prepared through whole-genome-PCR. A PCR-assisted procedure which combines protein-DNA interaction and affinity chromatography was designed to enrich Myc/Myn bound DNA. At least two genomic DNA fragments ob- tained exhibit specifical binding capacity to Myc/Myn complex but not to GST alone. Significance of the work and of the technique itself as well as identification of the DNAs are discussed.

Investigation on the Interface between Exons and Introns in the Genomic DNA of Arabidopsis thaliana in the Phase Space

In this study,three weight vectors L1,L2 and L3 were set.After calculating the probability of three bases in the exons or introns in the genomic DNA of Arabidopsis thaliana,64-dimensional vector P was obtained.Dot products of P vector and three weight vectors were the feature coordinates for the exons and introns in 3-dimensional phase space.The expression for the interface between the exons and the introns in the genomic DNA of Arabidopsis thaliana in 3-dimensional phase space was established,which could be used to distinguish the exons and the introns in the genomic DNA of Arabidopsis thaliana with an accuracy higher than85%in 3-dimensional phase space.

Pathogenic Effects of Cloned Genomic DNA of Porcine Circovirus-like Virus P1 on Neonatal Mice via Different Inoculation Routes

[Objective] The paper was to explore the pathogenicity of cloned genomic DNA of porcine circovirus-like virus Pl to neonatal mice via different inoculation routes （brain, liver and muscle）. [Method] Cloned genomic DNA of P1 was inoculated to neonatal mice via different routes of brain, liver and muscle. Tissues of heart, liver, spleen, lung, kidney and brain were taken from neonatal mice at 7, 14 and 21 d post inoculation, re- spectively. Pl in various tissues were qualitatively and quantitatively detected by using ordinary PCR and quantitative real-time PCR. Meanwhile, histopathological changes were analyzed. [Result] Pl was detected in neonatal mice inoculated through three different routes. The viral load of tis- sues at 7 d post inoculation was significantly higher than those at 14 and 21 d post inoculation. Moreover, muscle inoculation led to the highest viral load in all tissues of neonatal mice. [Conclusion] Pl infection caused different degrees of pathological damage to heart, liver, lung, kidney and brain in neonatal mice.

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.

Character of cell-free genomic DNA in embryo culture medium and the prospect of its clinical application in preimplantation genetic testing

There is increasing evidence that cell-free DNA (cfDNA) in spent culture media (SCM) can be amplified for genetic testing. Therefore, this paper reviews the characteristics of cfDNA, including its fragment size, amount, origin, as well as some factors affecting the success rate of its amplification, together to provide researchers with a more comprehensive perspective on embryonic cfDNA. The origin of cfDNA in SCM is complicated and poses challenges to the interpretation of genetic test results. Advanced molecular techniques should distinguish between embryonic and contaminated DNA to maximize the success rate of amplification and analysis. Recent data showed that the type of culture medium, assisted hatching or not, the type of amplification kit, and fresh or thawed embryos were not related to the success rate of amplification, but the length of culture time might affect the success rate. The longer culture time, the more cfDNA is available in the SCM. Then we focused on the concordance between trophectoderm (TE), inner cell mass, whole embryo, and embryonic cfDNA. Despite successful amplification, the concordance between TE and embryonic cfDNA was low. In summary, non-invasive genetic testing using SCM could represent a major advance in future single embryo selection, however, contamination and timing for media collection are key factors affecting the results, and current non-invasive cfDNA testing should not be directly applied to clinical practice. Further research is needed to improve the methods used for testing techniques and genetic analysis to achieve greater accuracy and trace its origins before it can be used in the clinics.

Methylome and transcriptome data integration reveals potential roles of DNA methylation and candidate biomarkers of cow Streptococcus uberis subclinical mastitis

Background:Mastitis caused by different pathogens including Streptococcus uberis(S.uberis)is responsible for huge economic losses to the dairy industry.In order to investigate the potential genetic and epigenetic regulatory mecha‑nisms of subclinical mastitis due to S.uberis,the DNA methylome(whole genome DNA methylation sequencing)and transcriptome(RNA sequencing)of milk somatic cells from cows with naturally occurring S.uberis subclinical mastitis and healthy control cows(n=3/group)were studied.Results:Globally,the DNA methylation levels of CpG sites were low in the promoters and first exons but high in inner exons and introns.The DNA methylation levels at the promoter,first exon and first intron regions were nega‑tively correlated with the expression level of genes at a whole‑genome‑wide scale.In general,DNA methylation level was lower in S.uberis‑positive group(SUG)than in the control group(CTG).A total of 174,342 differentially methylated cytosines(DMCs)(FDR<0.05)were identified between SUG and CTG,including 132,237,7412 and 34,693 DMCs in the context of CpG,CHG and CHH(H=A or T or C),respectively.Besides,101,612 methylation haplotype blocks(MHBs)were identified,including 451 MHBs that were significantly different(dMHB)between the two groups.A total of 2130 differentially expressed(DE)genes(1378 with up‑regulated and 752 with down‑regulated expression)were found in SUG.Integration of methylome and transcriptome data with MethGET program revealed 1623 genes with signifi‑cant changes in their methylation levels and/or gene expression changes(MetGDE genes,MethGET P‑value<0.001).Functional enrichment of genes harboring≥15 DMCs,DE genes and MetGDE genes suggest significant involvement of DNA methylation changes in the regulation of the host immune response to S.uberis infection,especially cytokine activities.Furthermore,discriminant correlation analysis with DIABLO method identified 26 candidate biomarkers,including 6 DE genes,15 CpG‑DMCs and 5 dMHBs that discriminated between SUG and CTG.Conclusion:The integration of methylome and transcriptome of milk somatic cells suggests the possible involve‑ment of DNA methylation changes in the regulation of the host immune response to subclinical mastitis due to S.uberis.The presented genetic and epigenetic biomarkers could contribute to the design of management strategies of subclinical mastitis and breeding for mastitis resistance.

Remarkably different results between two studies from North America on genomic mutations and sensitivity to DNA demethylating agents for myelodysplastic syndromes

Sekeres et al. （1） conducted a multicenter randomized, controlled trial to compare whether azacitidine-based combinations with lenalidomide or vorinostat produce superior overall response rates to azacitidine in the treatment of myelodysplastic syndromes （MDS）. In that trial, 224 patients with higher-risk MDS and 53 with chronic myelomonocytic leukemia （CMML） were enrolled and randomly assigned to the ＂azacitidine＂ group, ＂azacitidine plus lenalidomide＂ group or ＂azacitidine plus vorinostat＂ group. The researchers found that patients with MDS treated with azacitidine-based combinations had similar response rate to azacitidine monotherapy. Using genomic mutation analysis, they found that the overall response rate to azacitidine-based treatment was higher for patients with mutations in DNMT3A and lower for those with mutations in SRSF2. Whereas in another study, Welch et al. enrolled 26 patients with MDS and 90 with acute myeloid leukemia （AML） who were treated with decitabine, and they found that patients with TP53 mutations had a higher response rate, but not those with DNMT3A mutations （2）. We propose that this big discrepancy in the conclusions between the two studies might have been caused by the presence of many co-interacting factors, e.g. study aims, DNA demethylating agents, treatment protocols, and patient sources.

Analysis of genetic diversity for wild and captive green peafowl populations by random amplified polymorphic DNA technique

The genetic diversity of the populations for 14 wild green peafowls (Pavo muticus) and 18 captive green pea-fowls was investigated by using the technology of random amplified polymorphic DNA (RAPD). Totally 161 and 166 ampli-fied bands were obtained by using 23 arbitrary primers to amplify the genomic DNA of wild and captive green peafowls re-spectively. The results showed that the average relative genetic distance of the wild and captive green peafowls popula-tions was 0.0555 and 0.1355, respectively, and difference of the average relative genetic distances between the two popu-lations was 0.1635. The Shannon diversity index for the wild and captive green peafowl populations was 0.4348 and 1.0163, respectively, which means that there exists significant difference in genetic diversity between the two populations, and the genetic diversity of wild green peafowl was low. The two populations originated from two different families according to analysis by the UPGMA method. This research can provide the theoretical basis for supervising genealogies management of peafowl populations.

Higher frequency of Yq microdeletions in sperm DNA as compared to DNA isolated from blood

Aim： To determine if Yq microdeletion frequency and loci of deletion are similar in two tissues （blood and sperm） of different embryological origin. Methods： The present study included 52 infertile oligozoospermic cases. In each case, DNA was isolated from blood and sperms and polymerase chain reaction （PCR） microdeletion analysis was done from genomic DNA isolated from both the tissues. The PCR products were analyzed on a 1.8% agarose gel. PCR amplifications found to be negative were repeated at least three times to confirm the deletion of a given marker. Results： Only 1 case harbored microdeletion in blood DNA, whereas 4 cases harbored microdeletion in sperm DNA. Conclusion： The frequency of Yq microdeletions is higher in germ cells as compared to blood. As the majority of infertile couples opt for assisted reproduction procreation techniques （ART）, Yq microdeletion screening from germ cells is important to understand the genetic basis of infertility, to provide comprehensive counseling and most adapted therapeutics to the infertile couple.

A NOVEL HUMAN DNA SEQUENCE WITH TUMOR METASTASIS SUPPRESSIVE ACTIVITY

Objective: To isolate human tumor metastasis suppressive DNA sequence and to study the molecular mechanisms regulating tumor metastasis. Methods: A mouse lung adenocarcinoma cell clone 12 derived from its parent cell line LM2, which had been transduced with normal human genomic DNA, was previously reported. Compared with LM2, the metastatic potential of clone 12 was very much decreased. Clone 12 was used in this study to amplify the human DNA fragments by Inter Alu PCR technique. The human DNA fragments obtained were then transfected into LM2 cells and their malignant phenotype was tested in vitro and in vivo, and compared with that of the untransfected LM2 cells.Results Three human DNA fragments of 700, 500 and 300 bp were isolated. DNA sequencing revealed that the 700bp fragment does not show homology with hitherto reported genes and was accepted by the Genbank (pt712 U67835). In vitro proliferation and colony formation in soft agar of the 700 bp fragment-transfected LM2 cells were significantly inhibited as compared to the untransfected LM2 cells. Upon subcutaneous inoculation to syngeneic T739 mice, the 700bp-transfected LM2 cells grew more slowly and smaller tumors developed compared to the untransfected ones. Moreover, lung metastasis was not found in 6 of 10 mice inoculated with the 700bp-transfected LM2 cells, while it was found in 9 of 10 mice inoculated with the untransfected LM2 cells. The difference was statistically significant (P<0.001). The frequency of lymph node metastasis was also statistically different between the 2 groups of mice.Conclusion The newly isolated 700bp human DNA fragment may be a metastasis suppressor gene of malignant tumor.