Genomic DNA Extraction Kit Research Articles

Keberadaan Anisakis typica (Anisakidae) dari Ikan Tongkol dan Ikan Layang dari perairan Sulawesi Barat

Anisakis (L3-stage) is a parasitic nematode commonly found in marine fish or squid serve as intermediate or paratenic host. The purpose of this research was to identify Anisakis larvae using PCR-RFLP and sequencing methods. A total of 30 individuals of each species: bullet tuna (Auxis rochei) and Indian scad(Decapterus russeli) were examined for presence of Anisakis spp. Fish samples caught by fishermen around Mamuju waters were purchased at a fish market. The fish was necropsied and the organs: liver, intestine wall, heart and muscle were removed and put on separated petri dishes and examined for parasites under a stereo microscope. Anisakis found was cleaned and fixed in 70% alcohol. Initial identification was based on ventriculus shape and presence of mucron to distinguish Anisakis type I and Anisakis type II. All Anisakis found belong to Anisakis type I. Ten Anisakis type I were isolated, cleaned and stored in microfuge tubes containing 70% alcohol. The parasite gDNA was extracted using Wizard genomic DNA extraction and purification kit (Promega).Species of Anisakis was determined by PCR-RFLP at ITS1-5.8S-ITS2 region using two restriction enzymes: Taq I and Hinf I and PCR-sequencing in mtDNA cox2 region. Analysis by PCR-RFLP showed that all Anisakis type Iexamined was in the same pattern as Anisakis typica. Sequencing in mtDNAcox-2 region and the phylogenetic analysis showed that all samples were in the same cluster as A. typica. Based on PCR-RFLP and sequencinganalysis, all Anisakis found in this study belong to A. typica.

Analysis of haematological phenotype and mutation spectrum of δ-globin gene from Guangdong area in Chinese Han prenatal population

Objective: To analyze the genotype-phenotype correlations among southern Chinese Han prenatal population in Guangdong area with δ-globin gene mutation, so as to enrich the delta-thalassemia gene mutations data. Methods: A total of 33 cases were selected in 7 580 patients during prenatal thalassemia trait screening, from January 2012 to May 2015(including 10 males and 23 females, aged 22-48 years old). Complete blood cell count was performed on a XE 4000i automatic hemocyte analyzer. Hb, HbF and HbA2 were tested by high performance liquid chromatography (HPLC). Genomic DNA was extracted from whole blood samples using a whole blood genomic DNA extraction kit. Polymerase chain reaction (PCR) was used to amplify three different fragments corresponding to the exons and the regulatory sequences using three different couples of primers for the δ-globin gene. Results: Twenty one of the 33 samples were positive for the δ-globin gene defects. Four previously known mutations were detected: including 12 cases for -77(T>C)[HBD c. -127 (T>C)](57.14%), 4 cases for -30 (T>C)[HBD c. -80 (T>C)](19.05%), 1 case for codon 10 (-G) (HBD c. 31delG)(4.76%), and 1 case for HBD c. 244 C>T(4.76%). Three new δ-globin gene defects which had not yet been reported in database were detected, including 1 case for HBD c. 22_24delGAG(4.76%), 1 case for HBD c. 347 C>T(4.76%), and one case for HBD c. 349 C>G(4.76%). Conclusions: -77 (T>C) is the most common mutation in Chinese southern prenatal population. Three new HBD gene mutations are referred in this report, which provide the valuable information for genetic counseling and prenatal diagnosis in Guangdong area.

Effect of Source of DNA on the Quantitative Analysis of Genetically Engineered Traits Using Digital PCR and Real-Time PCR.

Seven commercially available DNA extraction kits were compared with a cetyltrimethylammonium bromide (CTAB) method to determine the suitability of the extracted DNA for RainDrop digital PCR (dPCR) and real-time PCR (RT-PCR) quantification of OXY235 canola, FP967 flax, and DP305423 soybean (spiked at the 0.1% level). For the kits, the highest amount of DNA extracted from a 0.2 g sample was obtained using OmniPrep for Plant for flax and DNeasy mericon Food for canola and soybean. For canola, DNA extracted with the Fast ID Genomic DNA Extraction Kit, FastDNA Spin Kit, GM Quicker 2, NucleoSpin Food, and DNeasy mericon Food was suitable for dPCR and RT-PCR. For flax, DNA extracted with Fast ID, FastDNA Spin Kit, OmniPrep for Plant, and NucleoSpin Food was suitable for RT-PCR. However, only Fast ID yielded DNA suitable for dPCR. For soybean, DNA extracted with five and six of the seven DNA extraction kits was suitable for dPCR and RT-PCR, respectively. Overall, Fast ID provided reliable results regardless of species or analysis method used. Canola, flax, and soybean DNA extracted with the CTAB method and then purified were suitable for both dPCR and RT-PCR. This is the first report showing the effect of different DNA extraction methods on the absolute quantification of genetically engineered traits using dPCR.

Species diversity and utilization of bamboo to support life’s the community of Karangwangi Village, Cidaun Sub-District of Cianjur, Indonesia

Abstract. Hailu HW, Sajidan, Pangastuti A, GreinerR. 2017. Isolation, characterization and 18S rDNA sequence analysis of phytase producing fungi from Indonesia, Indonesia. Biodiversitas 18: 10-14. Phytase breaks down the phytate portion of grains and oil seeds; thereby, releasing phosphorus and minerals for animals. The objectives of this research were to screen phytase-producing novel species of fungi; to conduct cultural and phylogenetic characterization; to perform 18S rDNA sequencing and BLAST analysis. Samples of fungi were collected from different sources, cultured on potato dextrose agar and potato dextrose broth. Phytase assay was conducted based on Vanadate-molybdate method. Three isolates with highest enzyme activity were selected for pH and temperature optimization. DNA was extracted by modifying plant DNA extraction and yeast genomic DNA extraction kits. The 18S rDNA gene was amplified and sequenced. The result indicated that optimum temperatures ranged from 60-75oC and the optimum pH for isolates from papaya (Angr), palm bark (Abark) and sugarcane soil field (Asoil) were 4.5, 3.0 and 4.5, respectively. Based on 18S rDNA sequence analysis the phytase producing fungal isolates Angr, Abark and Asoil were identical to Aspergillus niger, Beauveria felina and Nigrosabulum globosum, respectively.Keywords: 18S rDNA, BLAST, fungi, phytase, phylogeny

Prevalence of trypanosome infection in tsetse flies from Oji River and Emene axis of Enugu State, Nigeria: A preliminary report.

Introduction:Trypanosomes are protozoan parasites of vertebrates transmitted by blood-sucking tsetse fly. Trypanosomes remain a constant threat to the lives of humans and animals throughout large regions of Africa.Aims and Objectives:This study investigated the presence, prevalence, and species of trypanosome parasite in tsetse flies caught in two areas of no previous documented history of trypanosome infection.Materials and Methods:For this purpose, 63 and 77 nonterenal tsetse flies were collected from Oji River and Emene areas of Enugu State Nigeria, respectively. Genomic DNA was isolated from the whole tsetse fly using genomic DNA extraction kit. Identification and characterization of trypanosome were done using two approaches: the amplification of internal transcribed spacer 1 of ribosomal DNA and the use of primers specific to Trypanozoon.Results:In Oji River, of 63 tsetse flies collected, the identification of trypanosome parasite was done on 57 flies and 6 (10.71%) tsetse flies were infected with trypanosome parasite. Six flies were infected with Trypanosoma Congolense, 2 with Trypanosoma Vivax, and 1 with Trypanosoma brucei. Two mixed infections of T. vivax and T. congolense and 1 mixed infection of T. brucei and T. congolense was also identified. In Emene, of 77 tsetse flies collected, the identification of trypanosome parasite was done on 66 flies and 11 (16.6%) tsetse flies were infected with trypanosome parasite. Nine flies were infected with T. congolense, 2 with T. vivax, and 3 with T. brucei. Mixed infections identified include 2 mixed infections of T. brucei and T. congolense and 1 mixed infections of T. vivax and T. brucei. None of the subspecies of T. brucei were detected using species specific primers.Discussion:This study shows the parasitological evidence on the occurrence of animal African trypanosomiasis and also demonstrated that there is likely no active transmission of human African trypanosomiasis in the study areas.Conclusion:This study shows that there is likely no active transmission of human African trypanosomiasis going on in these localities since no human infective form of the parasite was detected.

Association between single nucleotide polymorphismsin human heat shock protein 70 gene and susceptibility to noise-induced hearing loss

Objective: To investigate the association between the single nucleotide polymorphisms (SNPs) at rs1043618, rs2075800, and rs2763979 in human heat shock protein 70 (HSP70) gene and susceptibility to noise-induced hearing loss (NIHL) . Methods: A case-control study was performed, and 5 934 workers exposed to noise in an iron and steel plant in Henan, China, who underwent physical examination from 2006 to 2015, were enrolled as study subjects. According to the criteria of binaural average high - frequency (3000, 4000, and 6000 Hz) hearing threshold≥40 dB (HL) and monauralaverage speech-frequency (500, 1000, 2000 Hz) hearing threshold≥26 dB (HL) on the basis of binauralhigh frequency loss measured by pure tone audiometry, as well as the exclusion of NIHL, a total of 286 workers were enrolled as hearing loss group; after the adjustment for sex, type of work, age (difference≤5 years) , and working years of noise exposure (difference ≤2 years) , 286 workers were enrolled as control group. A 2 ml blood genomic DNA extraction kit was used to perform DNA extraction for the peripheral blood samples, and a multiple SNP typing kit was used to determine the genotypes at the three loci in 572 samples. The association between the SNPs at the three loci and susceptibility to NIHL was analyzed. Results: In all workers, the equivalent sound level (L(Aeq)) of noise was 75.0~96.8 dB (A) . The hearing loss group had a significantly higher binauralhigh - frequencyhearing threshold than the control group (t=56.908, P<0.05) . With CC+TC genotype as control, TT genotype at rs2763979 in HSP70 gene was associated with the susceptibility to NIHL (OR=1.731, 95%CI 1.021-2.935) . In the group with cumulative noise exposure of 96 dB (A) ·year, TT genotype at rs2763979was associated with the susceptibility to NIHL (OR=5.694, 95%CI 1.256-25.817) . The rs1043618 and rs2075800 loci of HSP70 were not associated with the susceptibility to NIHL (both P>0.05) . After the adjustment for confounding factors including smoking and drinking, haplotype CCT was associated with the susceptibility to NIHL (OR=1.425, 95%CI 1.035-1.961) . Conclusion: TT genotype at rs2763979 of HSP70 gene and haplotype CCT are risk factors for NIHL.

A Simplified and Cheapest Method for the Diagnosis of Sickle Cell using Whole Blood PCR and RFLP in Nepal

Sickle cell anemia is a serious genetic health problem dominated in Tharu community of western Nepal. Molecular methods like PCR and RFLP are the best method to identify Sickle c ell anemia trait. Molecular analysis needed many steps and expensive chemicals and Kits. The aim of this research was to develop a simple and cheapest method to process from whole blood sample for the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) without the use of expensive reagents and Kits. In this study molecular identification of sickle cell traits is subjects using whole blood PCR and RFLP were carried out. Venous blood samples collected from 800 individuals were provided by Nepal Health Research Council (NHRC). All the obtained samples were frozen at –80°C, and then rapidly thawed at 37°C. Then the samples were transferred to 2 ml eppendrof tube and boiled for 10minutes in distilled water and centrifuged at 12000 rpm for 2 minute. The supernatant was then used directly for PCR and RFLP. For comparison, purified DNA from the QIAGEN genomic DNA extraction kit was used as control. PCR/RFLP results using the whole blood boiling method was qualitatively similar to DNA extracted by using commercial Kits. The research demonstrates that whole blood PCR and RFLP method is simple and cheaper way for molecular diagnosis of sickle cell traits in human.

식품으로부터 식중독 세균 검출을 위한 Real-time PCR에 적합한 DNA 추출 방법 비교

This study was conducted to find out the most suitable DNA isolation methods for PCR detection of foodborne pathogens. Four DNA isolation methods including Universal Genomic DNA Extraction Kit (TaKaRa), PrepMan Ultra (Applied Biosystems), boiling method and alkaline lysis method (w/PEG) were tested and compared. The Universal Genomic DNA Extraction kit (TaKaRa) was considered as the more efficient isolation method for Escherichia coli O157:H7 and Staphylococcus aureus in lettuce, fish and beef. Meanwhile to detect the foodborne pathogens directly from foods without enrichment, the four different buffers such as double-distilled water, saline, glycine-saline, glycine-saline with Tween-20 and beef extract were also evaluated. As a result, saline was more suitable buffer for E. coli O157:H7. And double-distilled water was more suitable buffer than saline for S. aureus, respectively

Molecular Characterization of Fasciola Spp. Isolated From the Gallbladder of Infected Cattle in Duhok Province, Kurdistan Region Iraq

Fascioliasis caused by the liver flukes of the genus Fasciola is considered as the most significant Trematodes infection of ruminants in both temperate and tropical countries. In the present study fifty adult Fasciola flukes (Platyhelminthes: Trematoda: Digenia) were collected from the bile ducts and gallbladder of infected bovine hosts (cattle) slaughtered at Duhok abattoir, Kurdistan region, Iraq. Genomic DNA extraction was performed using a Genomic DNA Extraction kit. ITS-1 and ITS-2 ribosomal DNA sequences have been used to characterize these liver flukes as a specific marker. The PCR products were separated by electrophoresis in 1.5% agarose gel, visualized by staining with ethidium bromide, and photographed. ITS-2 marker for F. hepatica was amplified successfully and the length of produced band for ITS-2 was 330 bp. The present study is the first trail for molecular characterization of F. hepatica in cattle in Duhok, Kurdistan Region, Iraq using the ITS-2 rDNA as a reliable genetic marker.

First Report of Sweet potato badnavirus A in China

HomePlant DiseaseVol. 100, No. 4First Report of Sweet potato badnavirus A in China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Sweet potato badnavirus A in ChinaY.-H. Qin, X.-C. Li, Z.-C. Zhang, Q. Qiao, D.-S. Zhang, Y.-J. Wang, Y.-T. Tian, and S. WangY.-H. QinSearch for more papers by this author, X.-C. LiSearch for more papers by this author, Z.-C. ZhangSearch for more papers by this author, Q. QiaoSearch for more papers by this author, D.-S. ZhangSearch for more papers by this author, Y.-J. WangSearch for more papers by this author, Y.-T. TianSearch for more papers by this author, and S. WangSearch for more papers by this authorAffiliationsAuthors and Affiliations Y.-H. Qin X.-C. Li Z.-C. Zhang Q. Qiao D.-S. Zhang Y.-J. Wang Y.-T. Tian S. Wang , Institute of Plant Protection, Henan Academy of Agricultural Sciences; and Henan Key Laboratory of Crop Pest Control; and IPM Key Laboratory in Southern Part of North China for Ministry of Agriculture, Zhengzhou, 450002, China. Published Online:5 Feb 2016https://doi.org/10.1094/PDIS-09-15-1081-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Sweet potato badnavirus A (SPBV-A) belongs to the genus Badnavirus of the family Caulimoviridae; thus far, this virus has been detected in sweet potato (Ipomoea batatas) in Peru (Kreuze et al. 2009) and Tanzania (Mbanzibwa et al. 2011). A survey was conducted from 2013 to 2015 to determine the incidence of SPBV-A in China. A total of 200 sweet potato samples showing vein clearing were collected from 16 provinces in China. Total nucleic acids were extracted from these samples using Universal Genomic DNA Extraction Kit Ver. 3.0 (TaKaRa, Dalian, China) in accordance with the manufacturer’s protocol. The partial polyprotein gene sequences of SPBV-A were amplified through PCR with the primer pairs BadnaBKF (5′-CAAATTAGGAGGCAGATAAATG-3′) and BadnaBSR (5′-GGTCTTCTTATGTTCCACCTT-3′) (Mbanzibwa et al. 2011). The expected 861-bp amplicons were amplified from 34 samples. PCR products were cloned into the pMD19-T vector (TaKaRa, Dalian, China) and then sequenced. The sequences were compared with those in GenBank using BLAST. Sequences in the 861-bp DNA fragment of the 34 samples (GenBank Accession Nos. KT448732 to KT448765) share 87.6 to 96.7% identity to the nucleotide sequence of the polyprotein gene of SPBV-A (Accession No. FJ560943); the result also revealed that the nucleotide sequences were 88.6 to 100% identical to each other. SPBV-A was detected in 34 of the 200 samples, and the positive rate was 17%. SPBV-A was distributed in Jilin, Shandong, Hebei, Shanxi, Shaanxi, Henan, Anhui, Guangxi, Fujian, Jiangsu, Chongqing, and Sichuan provinces in China. The total RNA of the 3 samples obtained from the positive sweet potato samples were randomly extracted from 0.1 g of fresh leaves by using the Total Plant RNA Extraction Miniprep System (Sangon, Shanghai, China) to confirm SPBV-A infection in China. Deep sequencing was conducted using Illumina Solexa 2000 (Beijing Berry Genomics Co., Ltd., Beijing). The pooled RNA (10 μg) was subjected to small RNA deep sequencing, and the results revealed 11,638,346 reads and 418,980,456 bases. The sequences were assembled with SOAPdenovo2 (http://soap.genomics.org.cn/soapdenovo.html) software with these settings: −k = 15, −d = 1, −R = 21, and −m = 21. The assembly generated gene fragments specific to 14 kinds of viruses, including Sweet potato feathery mottle virus, Sweet potato virus C, Sweet potato badnavirus B, Sweet potato badnavirus A, Sweet potato virus G, Sweet potato virus 2, Sweet potato leaf curl Georgia virus, Sweet potato chlorotic stunt virus, Sweet potato leaf curl virus, Sweet potato leaf curl Canary virus, Sweet potato leaf curl Bengal virus, Sweet potato leaf curl Lanzarote virus, Sweet potato leaf curl Uganda virus, and Sweet potato gold vein associated virus. The 370- and 358-bp SPBV-A genomic sequences were assembled from these three samples and then compared with the sequences of SPBV-A in GenBank. The finding also revealed nucleotide sequence identities of 87% and 89% to the SPBV-A sequence (Accession No. FJ560943), respectively, confirming the presence of SPBV-A in these three samples. Two sweet potato vine cuttings selected from the positive samples were grafted onto Ipomoea setosa, a universal plant indicator of sweet potato viruses (Schaefers and Terry 1976). At 3 to 4 weeks postinoculation, the graft-inoculated I. setosa plants exhibited leaf mosaic symptoms. The SPBV-A infection of the indicators was confirmed through PCR and sequencing. To our knowledge, this is the first report of SPBV-A on sweet potato in China. This study adds to the understanding of the presence and genetic diversity of this virus in China; further study into the damage and management strategy of this virus is required.

Molecular identification of Uncaria (Gouteng) through DNA barcoding.

BackgroundWhile DNA barcoding is an important technology for the authentication of the botanical origins of Chinese medicines, the suitable markers for DNA barcoding of the genus Uncaria have not been reported yet. This study aims to determine suitable markers for DNA barcoding of the genus Uncaria (Gouteng).MethodsGenomic DNA was extracted from the freshly dried leaves of Uncaria plants by a Bioteke’s Plant Genomic DNA Extraction Kit. Five candidate DNA barcode sites (ITS2, rbcL, psbA–trnH, ITS, and matK) were amplified by PCR with established primers. The purified PCR products were bidirectionally sequenced with appropriate amplification primers in an ABI-PRISM3730 instrument. The candidate DNA barcodes of 257 accessions of Uncaria in GenBank were aligned by ClustalW. Sequence assembly and consensus sequence generation were performed with CodonCode Aligner 3.7.1. The identification efficiency of the candidate DNA barcodes was evaluated with BLAST and nearest distance methods. The interspecific divergence and intraspecific variation were assessed by the Kimura 2-Parameter model. Genetic distances were computed with Molecular Evolutionary Genetics Analysis 6.0.ResultsThe accessions of the five candidate DNA barcodes from 11 of 12 species of Uncaria in China and four species from other countries were included in the analysis, while 54 of total accessions were submitted to GenBank. In a comparison of the interspecific genetic distances of the five candidate barcodes, psbA–trnH exhibited the highest interspecific divergence based on interspecific distance, theta prime, and minimum interspecific distance, followed by ITS2. The distribution of the interspecific distance of ITS2 and psbA–trnH was higher than the corresponding intraspecific distance. Additionally, psbA–trnH showed 95.9 % identification efficiency by both the BLAST and nearest distance methods regardless of species or genus level. ITS2 exhibited 92.2 % identification efficiency by the nearest distance method, but 87 % by the BLAST method.ConclusionWhile psbA–trnH and ITS2 (used alone) were applicable barcodes for species authentication of Uncaria, psbA–trnH was a more suitable barcode for authentication of Uncaria macrophylla.Electronic supplementary materialThe online version of this article (doi:10.1186/s13020-015-0072-7) contains supplementary material, which is available to authorized users.

Genetic variability among cattle breeds of Nigeria using thyroid hormone responsive spot 14 alpha gene (THRSPα) through polymerase chain reaction (PCR)

The genetic variability among three Nigerian indigenous cattle breeds-White Fulani (WF), Red Bororo (RB) and Sokoto Gudali (SG) was carried out using Thyroid Hormone Responsive Spot 14 Alpha Gene (THRSPα) through Polymerase Chain Reaction (PCR). A total of sixty-seven (67) cattle blood samples comprising 30 WF, 25 RB and 12 SG were used. The DNA were extracted from blood samples using Zymobead Genomic DNA extraction kit after which PCR was carried out using 50 ng template DNA, 1.0μM primer (THRSPα), 16μl Nucleasefree water in a BIONEER Accupower premix. Gel electrophoresis was carried out and the gels scored. Statistical analyses were carried out using GENEPOP, PAST, SPSS version 16 and Tools for Population Genetic Analysis (TFPGA) version 1.3. The allelic frequency ranged from 0.3600 to 0.6400 in A and B alleles, average heterozygosity ranged from 0.4608 to 0.4861. Lowest genetic distance of 0.0010 between WF and RB and highest genetic distance of 0.0058 between SG and RB were identified. Lowest genetic identity of 0.9942 between RB and SG and highest genetic identity of 0.9990 between WF and RB was also identified. Two (2) genetic population clusters were identified in the dendogram; WF and RB are in cluster 1 while SG is in cluster 2. Test of Hardy Weinberg equilibrium revealed variation in genotypic frequencies. These results therefore demonstrate variation among these three Nigerian indigenous cattle breeds which is attributed to the response of the breeds to various stimuli which also enhance their survivability and adaptation to their specific environments. Such variation can also be harnessed for conservation and improvement of our indigenous breeds through selection and breeding strategies.

Direct DNA quantification in cell lysates by fluorometric method in combination with an advanced chemometric model

Accurate quantification of genomic DNA is fundamental to many molecular analyses such as diagnostic assays and genotyping. Due to relatively low and varying extraction rates of DNA from cell lysates by genomic DNA extraction kits, it is desired to quantitatively determine DNA contents directly in cell lysates. However, the coexistence of scatterers (e.g., cell debris), absorbers, and intrinsic fluorophores in cell lysates prevents either UV–Vis spectrophotometric methods or fluorometric methods based on fluorescent dyes and ratiometric models to be directly applied to cell lysates. In this contribution, a novel fluorometric method based on an advanced chemometric model which can effectively eliminate the effects of scatterers and absorbers on fluorescence spectra of cell lysates was proposed for the direct quantification of DNA in lysates of CEM cells. Without cumbersome prior DNA extraction, the proposed method achieved quite stable and accurate DNA quantification with recovery rates in the range of 89%–115%, considerably better than that of either the fluorometric method based on the ratiometric model or the UV–Vis spectrophotometric method coupled with DNA extraction. The proposed method has the advantages of rapidness, simplicity, and high precision, and can be developed into a promising alternative for accurate quantification of genomic DNA.

A simple method to overcome the inhibitory effect of heparin on DNA amplification.

Genetic material from large patient cohorts is increasingly central to translational genetic research. However, patient blood samples are a finite resource and their supply and storage are often dictated by clinical and not research protocols. Our experience supports difficulty in amplifying DNA from blood stored in herparin; a scenario that other researchers may have or will encounter. This technical note describes a number of simple steps that enable successful PCR amplification. DNA was extracted using the Illustra Nucleon Genomic DNA Extraction Kit. PCR amplification was attempted using a number of commercially available PCR mastermixes. PCR DNA amplification failed using ReddyMix™ PCR Master Mix, Thermo-Start® (Thermo Scientific Inc. US) and ZymoTaq™ (Zymo research, US) PCR mastermixes, as demonstrated absence of products on gel electrophoresis. However, using the Invitrogen™ (Thermo Scientific Inc., US) Platinum® Taq DNA Polymerase, PCR products were identified on a 1% agarose gel for all samples. PCR products were cleaned with ExoSAP-IT® (Affymetrix Inc., US) and a sequencing reaction undertaken using a standard Big Dye protocol. Subsequent genotyping was successful for all samples for alleles at the CDH1 locus. From our experience a standard phenol/chloroform purification and using the Invitrogen™ Platinum® Taq has enabled the amplification of whole blood samples taken into lithium heparin and stored frozen for up to a month. This simple method may enable investigators to utilise blood taken in lithium heparin for DNA extraction and amplification.

Association of common filaggrin null mutations with atopy but not chronic rhinosinusitis

Association of common filaggrin null mutations with atopy but not chronic rhinosinusitis

Methylene tetrahydrofolate reductase genotypes frequencies: association with toxicity and response to methotrexate in rheumatoid arthritis patients.

The purpose of this study was to determine the genotype/allele frequencies of C677T and A1298C polymorphisms of methylenetetrahydrofolate (MTHFR) gene in Jordanian rheumatoid arthritis (RA) patients. In addition, the association between MTHFR gene C677T and A1298C polymorphisms and response to and toxicity of methotrexate (MTX) was evaluated. 159 adult rheumatoid arthritis ent Rheumatology Clinic at The Jordan University Hospital, between December, 2011 and April, 2012 were recruited into the study. Genomic DNA was extracted from leukocytes using Wizard Genomic DNA extraction Kit. The DNA extracts were amplified by polymerase chain reaction (PCR), and the PCR products were subjected to restriction enzymes to identify the C677T and A1298C genotypes. Genotype frequencies were identified and their relationship to some measures of MTX response and toxicity were evaluated. The 677 TT genotype frequency was higher in RA patients (15.1%) compared to the healthy control group (5.9%) (odds ratio 3.09, 95% confidence interval (CI) 1.39-6.87, p-value=0.0056). No such differences were seen with the A1298C genotypes. The frequencies of 677T and 1298C variant alleles were 0.346 and 0.296, respectively. None of the change-in-disease-activity measurements in MTX-treated patients has a significant association with the various genotypes. There was no significant association between A1298C genotypes and "œany MTX toxicity", but there was an association between C677T polymorphism and "œany MTX toxicity" (p=0.037). In addition, there was no association between both SNPs and specific MTX adverse effects. However, some haplotypes or combinations of the C677T and A1298C genotypes were associated with certain MTX side effects. More data from a larger number of RA patients are needed to evaluate the role of pharmacogenetic studies of MTHFR gene in predicting MTX response and toxicity.

First Report of Septoria apocyni Causing Spot Blight on the Species of Apocynum venetum and Poacynum pictum in China.

The species of Apocynum venetum and Poacynum pictum grow widely from the middle to northwestern regions of China. During the summers of 2011 to 2013, a spot blight was found in wild and cultivated both species in Altay Prefecture of the Xinjiang Uygur Autonomous Region, China. The spot blight caused leaf yellowing and leaf drop, and serious damage to plant phloem. Lesions were circular to irregular, and the diameter of lesions on A. venetum and P. pictum was 1.84 to 6.84 × 1.23 to 4.24 mm and 2.05 to 7.09 × 1.46 to 5.65 mm, respectively. Pycnidia were 70 to 115 × 52 to 120 μm, scattered, spherical, buried, and had a brown hard shell with a prominent ostiole. Conidia were colorless, needle-shaped, or linear. The conidia base was obtuse, containing 3 to 5 indistinct septa, 46.3 to 110.3 × 2 to 2.5 μm. Fungal cultures were obtained by cutting 1-cm-long infected leaf pieces from the margins of the lesions following routine surface sterilizing procedures. The sections were placed on potato dextrose agar (PDA) in petri dishes and incubated at 23°C for 4 weeks (4). Hyphae had septa, the aerial and base mycelium was white and rufous, and the back of the colony was sunken and cracked after 2 weeks, but no spore was observed. To verify the identity, total DNA was extracted directly from fungal mycelium with a UNIQ-10 fungal genomic DNA extraction kit (Sangon Biotech, Shanghai, China) and PCR amplification performed with primers ITS1/ITS4 (3). A 512-bp PCR product was sequenced and contrasted with GenBank sequences using BLAST, which revealed 99% identity with Septoria sp. (GenBank Accession No. KC134322.1). To confirm pathogenicity, A. venetum and P. pictum were planted in pots and grown in a greenhouse. After 6 weeks of growth, plants were inoculated by spraying a mycelial suspension onto the foliage while control plants received a similar application of sterilized distilled water. Five pots (3 plants per pot) were used for each treatment. The pots were then placed on plates filled with tap water and covered with Plexiglas hoods in the greenhouse at 20 to 25°C. Lesions began to appear 6 to 7 days after inoculation with the mycelial suspension, whereas control plants remained healthy. The average disease incidence was 19.3%. The symptoms and morphology were similar to Septoria apocyni in Teterevnikova (2). It was determined that spot blight of A. venetum and P. pictum was caused by S. apocyni based on morphological comparison. There is one relevant literature report of spot blight on A. venetum and P. pictum in China, but without any details of the pathogenicity or morphology of the pathogen (1). We believe that this is the first report of S. apocyni occurring on the species of A. venetum and P. pictum in China.

Essential role of polymorphism of Gab1, EGFR, and EGF for the susceptibility of biliary tract cancer.

Cholangiocarcinoma is a malignant neoplasm arising from the epithelial cells lining the biliary ducts and its occurrence can be anatomically classified as within the liver (intrahepatic) or outside the liver (extrahepatic). Extrahepatic cholangiocarcinoma, which can be called as biliary tract cancer (BTC), is the most common form of this malignancy, and its etiology is still unclear. In this study, we tried to elucidate the complicated association between receptor tyrosine kinase (RTK) gene polymorphisms and susceptibility of BTC by analyzing frequency distribution of genotypes and alleles of GRB2-associated-binding protein 1 (Gab1), endothelial growth factor receptor (EGFR), and endothelial growth factor (EGF) and identified potential risk of BTC for people carrying specific genotype of Gab1 and EGFR. Two hundred twenty-five and 300 patients with BTC and cholelithiasis (gallstone (GS)), respectively, and 300 controls matched by age, sex, and ethnicity with patients were recruited from Shengjing Hospital of China Medical University from January 2008 to July 2011 with informed consents. Genomic DNA of BTC group was extracted and purified from formalin-fixed, paraffin-embedded tumor tissue sections using QiAamp DNA FFPE Tissue kit. For GS group and controls, DNA was extracted from peripheral blood leukocytes using genomic DNA extraction kit from Aid Lab. Target genes of RTK family were identified from National Center of Biotechnology Information (NCBI) SNP database and Japanese Single Nucleotide Polymorphisms (JSNP) database. Frequency distribution of genotypes and alleles was analyzed using HapMap Project database. All of the statistical analysis was conducted with SPSS 13.0 software. Eight loci were identified for Gab1 (4), EGFR (3), and EGF (1) as the target single-nucleotide polymorphisms (SNPs) for the association of gene polymorphisms and BTC. A/A genotype and A allele of rs3805246 in Gab1 and G/G genotype and G allele of rs2017000 in EGFR were significantly higher in BTC group than in GS group or controls. After controlling for BMI, age, gender, and smoking habit, patients with "A/A + G/A" had 2.154 times odds to have BTC; as for patients with "A/A" only, they still had 1.976 times odds to have BTC. In the rs2017000 of EGFR, patients with "G/G + G/A" had 1.772 times odds to have BTC, and patients with "G/G" only had 1.530 times odds to have BTC. Furthermore, patients with A/A in rs3805246 and G/G in rs2017000 simultaneously had 1.620 times chance to have BTC than people with other genotypes. This study explored the independent potential effect of EGFR signaling transduction pathway and its downstream element Gab1 and the gene-gene interaction on the disease mechanism of BTC in the perspective of genetics and molecular epidemiology.

Http://www.omicsonline.org/open-access/overcoming-pseudomonas-aeruginosa-resistance-caused-by-glycocalyx-with-tobracef-1948-5948.1000145.php?aid=25925

Oysters are filter feeders that bioaccumulate bacteria in water while feeding. To evaluate the bacterial genomic DNA extracted from retail oyster tissues, including the gills and digestive glands, four isolation methods were used. Genomic DNA extraction was performed using the Allmag™ Blood Genomic DNA (Allrun, Shanghai, China), the MiniBEST Bacterial Genomic DNA Extraction kits (Takara, Dalian, China), and the phenol-chloroform and boiling lysis methods. The concentration of the genomic DNA was measured using a spectrophotometer. The purity of the genomic DNA was evaluated by PCR amplification of 16S rDNA followed by determining the cloning efficiency of the amplicon into the pMD19-T vector. Furthermore, the bacterial DNA quality was also evaluated by PCR assays using a pair of species-specific primers for Vibrio parahaemolyticus. Our results showed that the two commercial kits produced the highest purity of DNA, but with the lowest yields. The phenol-chloroform method produced the highest yield although it was time-consuming. The boiling lysis method was simple and cost effective; however, it was only suitable to isolate genomic DNA from bacteria present in retail samples following an enrichment step. The two commercial kits were good candidates for genomic DNA extraction from retail oyster tissues without enrichment.

Production of CMAH Knockout Preimplantation Embryos Derived From Immortalized Porcine Cells Via TALE Nucleases

Although noncancerous immortalized cell lines have been developed by introducing genes into human and murine somatic cells, such cell lines have not been available in large domesticated animals like pigs. For immortalizing porcine cells, primary porcine fetal fibroblasts were isolated and cultured using the human telomerase reverse transcriptase (hTERT) gene. After selecting cells with neomycin for 2 weeks, outgrowing colonized cells were picked up and subcultured for expansion. Immortalized cells were cultured for more than 9 months without changing their doubling time (~24 hours) or their diameter (< 20 µm) while control cells became replicatively senescent during the same period. Even a single cell expanded to confluence in 100 mm dishes. Furthermore, to knockout the CMAH gene, designed plasmids encoding a transcription activator-like effector nuclease (TALENs) pairs were transfected into the immortalized cells. Each single colony was analyzed by the mutation-sensitive T7 endonuclease I assay, fluorescent PCR, and dideoxy sequencing to obtain three independent clonal populations of cells that contained biallelic modifications. One CMAH knockout clone was chosen and used for somatic cell nuclear transfer. Cloned embryos developed to the blastocyst stage. In conclusion, we demonstrated that immortalized porcine fibroblasts were successfully established using the human hTERT gene, and the TALENs enabled biallelic gene disruptions in these immortalized cells.