Preparation and evaluation of reference of enterohemorrhagic Escherichia coli strains and genomic DNA / 中国生物制品学杂志

@#Objective To screen enterohemorrhagic Escherichia coli(EHEC) strain and declare it as a standard strain of China Medical Bacterial Species Conservation and Management Center(CMCC).On the base,to prepare strain reference and genomic DNA reference of EHECand declare them as national drug reference with independent intellectual property rights in our country.Methods According to GB4789.6-2016 National Food Safety Standards-Food Microbiology TestDiarrheagenic Escherichia coil Test,the EHEC strain was screened from 160 Escherichia coli strains from patients with diarrhea and declared as a standard strain of CMCC according to management regulations.EHEC bacterial solution and genomic DNA solution were prepared,and freeze-drying technology was used to prepare 600 strain(10~3 CFU/sample) and genomic DNA(20 ng/sample) samples respectively.20 strain and 20 genomic DNA samples were randomly selected for uniformity test.Samples storing at 25 ℃ and 37 ℃ for 1,3,5 and 7 d were taken respectively to test the transportation stability.Then the samples were tested for the short-term storage stability by storing at 4 ℃ for 7,14 and 28 d,and for the long-term storage stability by storing at 20 ℃ for 14,28 and 60 d.Three laboratories were organized for collaborative calibration.20 food products were chosen as the substrate to evaluate the application effect of strain samples.Results The one EHEC strain selected from 160 Escherichia coli strains from patients was finally declared as the CMCC(B) 43207standard strain.In the uniformity test,F_(strainsample)=0.662 0.05,and eae,stxl and stx2 of 20genomic samples were all positive.After storage at 25 and 37 ℃ for 7 d,-20 ℃ for 60 d and 4 ℃ for 28 d,the viable bacteria content of the strain samples was still 103 CFU/sample,and eae,stxl and stx2 of the genomic samples were positive.EHEC strains and genomic DNA samples selected randomly were identified as EHEC by three laboratories,the viable bacteria content was 10~3 CFU/sample,and eae, stxl and stx2 were all positive.It was detected in 20 kinds of food substrates after adding samples,but not in the background control.EHEC strain and the genomic DNA sample were included in drug standard materials in our country,numbered 80024 and 80056,respectively.Conclusion The prepared EHEC strain and genomic DNA standard materials with independent intellectual property rights can improve the timeliness of EHEC testing and make up for the gap in our country.

Preliminary study of familial nonsyndromic tooth agenesis caused by ectodysplasin A mutation / 口腔疾病防治

Objective@# To explore the pathogenic genes in a Chinese family affected by nonsyndromic tooth agenesis so as to study the pathogenesis of oligodontia.@*Methods @# Hospital ethical approval and informed consent of the patients and family members were obtained. Clinical data of the proband and close family members were collected, peripheral venous blood was collected, and DNA was extracted. Gene sequencing was performed through whole-exome sequencing, and then the screened pathogenic genes were verified by Sanger sequencing. The three-dimensional structure of the mutant proteins was analyzed and compared with the wild-type using bioinformatics tools.@*Results@#The two patients with congenital majority tooth loss in this family were cousins, and there were no other patients with congenital majority tooth loss in the family. Besides congenital multiple tooth loss, the two patients had no obvious hair abnormalities, finger/toe abnormalities, sweating abnormalities or other abnormal manifestations of ectodermal tissue. We found a mutant gene that in this family by carrying out gene sequencing of the patients and their close family members. A novel EDA (ectodysplasin A) missense mutation c.983C>T (p. Pro328Leu) was identified, which changed the encoded amino acid from proline (Pro) to leucine (Leu). Analysis of the mutation site showed that the site was highly conserved, and three-dimensional structure modeling also found that it changed the structure of EDA. @* Conclusion@#A novel EDA missense variant (c.983C>T, p.Pro328Leu) was first identified in a Chinese family with nonsyndromic tooth agenesis, extending the mutation spectrum of the EDA gene.

Efecto citotóxico y genotóxico in vitro del extracto crudo y etanólico del rizoma de Curcuma longa L / In vitro cytotoxic and genotoxic effect of the crude and ethanolic extract from the rhizome of Curcuma longa L

RESUMEN Objetivos: Determinar el efecto citotóxico y genotóxico in vitro del extracto crudo y etanólico del rizoma de Curcuma longa L. Materiales y métodos: El efecto citotóxico fue evaluado utilizando líneas celulares DU-145, HT-29, 3T3 BALB/c. Se hallaron los porcentajes de crecimiento en 48 horas y se determinó la concentración inhibitoria 50 (CI50). El efecto genotóxico en el ADN genómico humano se determinó mediante el método Tomasevich. Resultados: El extracto crudo produjo una CI50 de 12,98 ± 0,21 μg/mL para la línea celular tumoral HT-29, que es inferior a DU-145 con una CI50 de 36,77 ± 9,12 μg/mL; el extracto etanólico presentó una CI50 de 13,24 ± 0,77 y 20,54 ± 2,58 µg/mL para ambas líneas celulares, respectivamente; el compuesto estándar curcumina presentó una CI50 de 3,96 ± 0,60 y 13,94 ± 2,79 μg/mL, respectivamente. El extracto crudo a concentraciones de 50 y 100 mg/mL fragmentó entre el 40% a 95% de ADN genómico humano; mientras que, a 200 mg/mL, la fragmentación fue mayor al 95%. El extracto etanólico a todas las concentraciones no fragmentó el ADN. La curcumina a 200 mg/mL fragmentó menos del 5% de ADN genómico humano. Conclusiones: Los extractos crudo y etanólico de Curcuma longa L. demuestran efecto citotóxico in vitro diferencial para la línea celular tumoral humana DU-145 y HT29 semejante al compuesto estándar curcumina. El extracto crudo de Curcuma longa L. presenta una potente actividad genotóxica in vitro frente al ADN genómico humano, esta actividad está ausente en el extracto etanólico.

ABSTRACT Objectives: To determine the in vitro cytotoxic and genotoxic effect of the crude and ethanolic extract from the Curcuma longa L. rhizome. Materials and methods: The cytotoxic effect was evaluated using DU-145, HT-29, 3T3 BALB/c cell lines. The growth percentages in 48 hours; and the half maximal inhibitory concentration (IC50) were determined. The genotoxic effect on human genomic DNA was determined using the Tomasevich method. Results: Crude extract produced an IC50 of 12.98 ± 0.21 μg/mL for the HT-29 tumor cell line, which is lower than the value obtained for DU-145, with an IC50 of 36.77 ± 9.12 μg/mL. The ethanolic extract presented an IC50 of 13.24 ± 0.77 and 20.54 ± 2.58 μg/mL for both cell lines, respectively; the curcumin standard compound presented an IC50 of 3.96 ± 0.60 and 13.94 ± 2.79 μg/mL, respectively. Crude extract concentrations of 50 and 100 mg/mL fragmented between 40% to 95% of human genomic DNA; while at 200 mg/mL, fragmentation was greater than 95%. The ethanolic extract at all concentrations did not fragment the DNA. Curcumin at 200 mg/mL fragmented less than 5% of human genomic DNA. Conclusions: The crude and ethanolic extracts of Curcuma longa L. demonstrate different in vitro cytotoxic effects for the human tumor cell lines DU-145 and HT-29; similar to the standard curcumin compound. The crude extract of Curcuma longa L. shows a potent genotoxic in vitro activity against human genomic DNA; this type of effect is not produced by the ethanolic extract.

Expression and characterization of a novel cold-adapted lipase from psychrotolerant bacteria Psychrobacter sp. S1B

@#Aim: This study aimed to isolate, express and characterize the lipase derived Psychrobacter sp. S1B in Escherichia coli expression system. Methodology and results: Exploration towards S1B lipase characteristic was conducted where shotgun cloning method was applied to obtain lipase encoded gene and E. coli expression system through pET28a was used to overexpress S1B lipase. Lipase activity was measured by using p-nitrophenol method. The S1B lipase gene is 1005 bp in length with molecular weight of 46 kDa, optimum pH was 10.0, showed hydrolytic activity preference toward p-nitrophenyl caprylate (C8) and p-nitrophenyl hexanoate (C6) substrates (C6 < C8). The best temperature for S1B lipase activity was at 30 °C while exhibited high activity at lower temperature (10-25 °C) with above 90% of maximum activity, therefore it is classified as cold adaptive lipase. In addition, S1B lipase showed stability against various metal ions, including Cu2+ and Zn2+ which commonly act as inhibitors of lipases derived from Psychrobacter species. Moreover, S1B lipase exhibited great tolerance against up to 50% (v/v) hexane and some non-ionic detergents such as 1% (v/v) DMSO and 1% (v/v) Triton X-100. Conclusion, significance and impact of study: The study proposes a novel cold-adapted lipase which has potential as a biocatalyst for synthesis caprylic acid ester

Role of Angiotensin Converting Enzyme (ACE) Gene Polymorphism in Breast Cancer among North Indian Population

Background: The most common cause of cancer related death among women in the world is Breast cancer (BCa). Almost Every year, approximately 1,300,000 cases and 450,000 deaths are related with Carcinoma of Breast are reported worldwide. The incidence of invasive Carcinoma of Breast and mortality in American women in 2017 was 252,710 and 40,610 respectively as quoted by a study. According to latest survey conducted by the Indian Council of Medical Research (ICMR) in India, there were an estimated 150,000 new cases of Carcinoma of Breast in the year 2016. The rise in both Carcinoma of Breast incidence and mortality, therefore, necessitates an examination of risk factors associated with this disease. Molecular subtypes-based classification system characterized by the presence or absence of immunohistochemical expressions like Progesterone receptor (PR), Estrogen receptor (ER), and Human epidermal growth factor receptor 2 (HER2) may show certain limitations. The gene encoding ACE (Angiotensin Converting Enzyme,) in humans is located in the chromosome 17 (17q23), consisting of 26 exons and 25 introns and spanning 21 kb. ACE is a zinc dependent dipeptidyl carboxypeptidase which catalyzes conversion of inactive decapeptide Angiotensin I (Ang I) to active octapeptide Ang II . Ang II mediates physiological effects by binding to two subtypes of the receptors, AGTR1 and Angiotensin II receptor type II (AGTR2), which belongs to superfamily of G-protein-coupled receptors (GPCRs).So, keeping all these physiological effects in mind, this study was conducted to see the role of ACE gene in carcinoma of breast. Methods: From confirm and control cases 3.0 ml of venous blood from each study subject was collected in an EDTA vial. Genomic DNA was extracted by phenol-chloroform method. The genotyping was performed by using PCR (Polymerase Chain reaction), using gene-specific primers. The resulting PCR products were separated on 2% agarose gels using ethidium bromide stain and visualized under UV light. The clinicopathologic parameters of breast cancer patients were obtained from medical records. Results: Of the 10 patients, 3 (30%) had Deletion/deletion genotype DD, 6 (60%) had ID, and 1 (10%) had II genotypes. In control subjects, 2 (20%) had DD, 6 (60%) had ID, and 2 (20%) had II genotypes. Conclusion: The results showed no significant association of ACE gene polymorphism with breast cancer (p>0.05). There is a necessity to conduct large-scale studies with adequate methodological quality and larger sample size in order to come to a definitive conclusion.

Identification of Amelogenin Gene on Burnt Teeth Samples through Nested Polymerase Chain Reaction Amplification for Sex Identification / Jurnal Sains Kesihatan Malaysia

@#Sex determination is one of the basic components in victim identification. This study aims to ascertain the sex of an individual from burnt teeth samples exposed at different temperature and time through nested polymerase chain reaction (PCR) on the amelogenin (AMEL) sex marker, to calculate the specificity and sensitivity, and to compare with previous relevant studies. A total of 17 teeth samples was subjected to burning at different temperatures ranging from 100°C to 500°C, at 2 to 10 minutes. The whole tooth was used for deoxyribonucleic acid (DNA) extraction by phenol-chloroform method. All samples were quantified for DNA concentration and then analyzed with nested PCR using two pairs of AMEL primer and results of sex typing were recorded. Out of 17 samples, genomic DNA extracted from 6 samples have concentrations ranging from 27.3 – 130.6 ng/µL. Nested PCR could amplify 16 samples for AMEL gene. Sex typing using AMEL gene showed 76.47% accuracy. Sensitivity of AMEL primer was increased from 6.67% to 63.64% using nested PCR technique; specificity of both external and internal primer was reported at 100%. Nested PCR of AMEL gene proved to be a suitable method for unequivocal determination of sex from degraded DNA samples.

A Rapid Microwave Method for Isolation of Genomic DNA and Identification of White Rot Fungi

A rapid, effective and efficient method to identify the innumerable white rot fungal strains is of utmost importance. Mycelia of the unknown as well as know isolates of WRF, after alternative washing with TE buffer and sterile water, were suspended in TE buffer. Fungi in solution were then exposed to microwave. The crude extract contained genomic DNA which was extracted and amplified using ITS primers for further identification. Based on sequencing results the identity of known cultures was confirmed, while the unknown cultures were identified as Clitopilus scyphoides (AGUM004, BankIt2098576 MH172163); Ganoderma rasinaceum (AGUM007, BankIt2098576 MH172163); Schizophyllum sp (KONA001 BankIt2098576 MH172164; AGUM011 BankIt2098576 MH172165 and AGUM021 BankIt2098576 MH172166 respectively), Coprinellus disseminatus (BANG001, BankIt2098576 MH172167) and Lentinus squarrosulus (TAMI004, BankIt2098576 MH172167). The microwave method described for isolating quality DNA of WRF without further purification steps proved a novel method requiring less than ten minutes and minimized the chances of the presence of PCR inhibitors.

Optimization of SRAP-PCR reaction systems for Curcuma wenyujin / 中草药

Objective: To research on the most appropriate SRAP-PCR amplication system of Curcuma wenyujin. Methods: The concentration of Mg2+, dNTP, template DNA, primer, and Taq polymerase was optimized by amplifying technology and method of PCR based on isolating genomic DNA from C. wenyujin. Results: The best amplification system for C. wenyujin was as follows: total reaction volume of 25 μL, including Mg2+ (25 mmol/L) 2.0 μL, Taq polymerase(5 U/μL)0.4 μL, random primer (20 μmol/L) 2 μL, template DNA (5 ng/μL) 2.0 μL, dNTP (25 mmol/L) 2.0 μL, 10 × PCR buffer 2.5 μL. Conclusion: The stripes of amplification products are clearness, high brightness, and good reproducibility. It indicates that the reaction system and reaction procedures which are confirmed in this experiment are applied to the SRAP molecular markers in C. wenyujin, based for the genetic diversity of C. wenyujin.

Establishment of TaqMan probe real-time PCR for detection of Cryptococcus neoformans / 国际检验医学杂志

Objective To establish TaqMan probe real‐time Fluorescent Quantitative PCR in detecting Cryptococcus neoformans genomic DNA ,and to provide important method for detection of cryptococcal meningitis .Methods According to the Cryptococcus neoformans ITS‐rDNA sequences obtained from NCBI ,specific primers and probe were designed based on the conserved sequences , a specific 114 bp fragment was amplified by primers and probe ,then recombinant plasmid was constructed .Eight different concen‐trations from 1 .42 × 10 copy/μL to 1 .42 × 10 copy/μL were used as templates by 2 μL .In the optimum reaction condition ,the sen‐sitivity ,specificity and repeatability were evaluated and standard curve was established .15 clinical cryptococcal meningitis strains i‐solated from clinical diagnosis patients were detected .Results The real‐time PCR showed high sensitivity and specificity and was a‐ble to detect 2 .84 × 102 copies of plasmid DNA .The detection sensitivity was 2 .84 × 102 copies plasmid DNA by real‐time PCR ,no amplification curve was detected with human genomic DNA ,other fungus ,bacterias and viruses .The CV of inter‐assay were 2 .86% ,1 .48% and 1 .36% respectively with excellent reproducibility .Fifteen clinical isolated strains could be detected accurately . Conclusion A method of detection of Cryptococcus neoformans DNA by real‐time PCR is established successfully with high sensi‐tivity and specificity ,and the results are stable ,could diagnose cryptococcal meningitis rapidly and early .

Catostylus tagi: partial rDNA sequencing and characterisation of nematocyte structures using two improvements in jellyfish sample preparation

Background More than 200 Scyphozoa species have been described, but few have been properly studied regarding their chemical and genetic characteristics.Catostylus tagi, an edible Scyphozoa and the sole European Catostylidae, occurs in summer at Tagus and Sado estuaries. Neither a systematic comparison between the two Catostyluscommunities nor a chemical approach on their nematocytes had been carried out yet.Methods In order to achieve these purposes, optimisation of DNA extraction and of histochemical staining procedures were developed.Catostylus specimens from Tagus and Sado estuaries were compared by ribosomal 18S, 28S, and ITS1 partial sequencing. The morphochemistry of nematocytes was studied by optical and electronic microscopy.Results Macroscopic and molecular results indicated that both communities belong to the same species, C. tagi. The hematoxylin and eosin staining allowed the visualisation of nematocyst genesis and indicated a basic character for the macromolecules on the shaft of euryteles and on the tubule of isorhizae and birhopaloids. By Massons trichrome procedure, the basic properties of the tubules were confirmed and a collagenous profile for the toxins was suggested. Results of the alcian blue staining showed that the outer membrane of nematocyte may consist of macromolecules with acidic polysaccharides, consistent with NOWA and nematogalectin glycoproteins detected in Hydra, but also with poly-gamma-glutamate complex, chitin-like polysaccharides and hyaluronic acids. Through the von Kossa assays, calcium was detected; its position suggested interactions with polysaccharides of the membrane, with proteins of the contractile system or with both.Conclusions The optimisation of sample preparation for DNA extraction may facilitate further studies on little known jellyfish species. The improvement of the smear procedure simplified the use of stained reactions in zooplankton. Moreover, it was shown that good slide images might be acquired manually. The development of specific reactions, with traditional dyes and others, can give important contributions to clarify the chemical nature of the components of nematocytes. The characterisation of nematocyst toxins by staining tests is a goal to achieve.

Catostylus tagi: partial rDNA sequencing and characterisation of nematocyte structures using two improvements in jellyfish sample preparation

Background More than 200 Scyphozoa species have been described, but few have been properly studied regarding their chemical and genetic characteristics.Catostylus tagi, an edible Scyphozoa and the sole European Catostylidae, occurs in summer at Tagus and Sado estuaries. Neither a systematic comparison between the two Catostyluscommunities nor a chemical approach on their nematocytes had been carried out yet.Methods In order to achieve these purposes, optimisation of DNA extraction and of histochemical staining procedures were developed.Catostylus specimens from Tagus and Sado estuaries were compared by ribosomal 18S, 28S, and ITS1 partial sequencing. The morphochemistry of nematocytes was studied by optical and electronic microscopy.Results Macroscopic and molecular results indicated that both communities belong to the same species, C. tagi. The hematoxylin and eosin staining allowed the visualisation of nematocyst genesis and indicated a basic character for the macromolecules on the shaft of euryteles and on the tubule of isorhizae and birhopaloids. By Masson's trichrome procedure, the basic properties of the tubules were confirmed and a collagenous profile for the toxins was suggested. Results of the alcian blue staining showed that the outer membrane of nematocyte may consist of macromolecules with acidic polysaccharides, consistent with NOWA and nematogalectin glycoproteins detected in Hydra, but also with poly-gamma-glutamate complex, chitin-like polysaccharides and hyaluronic acids. Through the von Kossa assays, calcium was detected; its position suggested interactions with polysaccharides of the membrane, with proteins of the contractile system or with both.Conclusions The optimisation of sample preparation for DNA extraction may facilitate further studies on little known jellyfish species. The improvement of the smear procedure simplified the use of stained reactions in zooplankton. Moreover, it was shown that good slide images might be acquired manually. The development of specific reactions, with traditional dyes and others, can give important contributions to clarify the chemical nature of the components of nematocytes. The characterisation of nematocyst toxins by staining tests is a goal to achieve.(AU)

Comparison of genomic DNA content of liver and spleen tissue for different age of elderly / 局解手术学杂志

Objective To investigate the change of genomic DNA of liver and spleen tissue for different age of the elderly,and provide the experimental data for aging-related research. Methods 35 livers and 33 spleens of autopsied samples preserved in refrigerator at-80 ℃ were divided into 3 groups according to age:age 65y to 79y,age 80y to 89y,age≥90y. The content of DNA in liver and spleen was determined by ultraviolet absorbent method. Results Compaired with age 80y to 89y (0. 310 ± 0. 286)mg/mL,the content of DNA in liver was significant higher at age 65y to 79y (1.464 ±0.488)mg/mL and age ≥90y(1.147 ±0.333)mg/mL(P<0.05);Compared with age 80y to 89y(0. 938 ± 0. 589)mg/mL,the content of DNA in spleen was significant higher at age 65y to 79y(1. 723 ± 0. 726)mg/mL and age≥90y(1. 688 ± 0. 963)mg/mL(P<0. 05). The content of DNA was significant lower in liver (0. 856 ± 0. 658)mg/mL than that in spleen (1. 414 ± 0. 852)mg/mL. Conclusion The content of DNA in human liver and spleen tissue may be decrease along with aging. The content of DNA in the group at age≥90y may be increase. There were some differences between different viscera tissue in content of DNA.

Development of a thalassemia gene diagnosis method for dried blood spots / 国际检验医学杂志

Objective To devolope a method for extracting DNA from dried blood spots (DBS)and optimizing the operating procedure,which could be applied to clinical gene diagnosis of thalassemia.And the cross contamination of DBS punching and the storage stability of DBS were studied.Methods A total of 1 50 blood specimens were collected,and DBS were prepared.Circles (3 mm in diameter)were punched in the DBS,and eluted with lysis buffer.The eluting method and operating procedure were opti-mized.Genomic DNA extracted from the elution solution by magnetic beads,and were performed thalassemia gene test.Finally jud-ging whether the results of DBS and whole blood were consistent.Two methods of thalassemia gene test were used in DBS and the compatibility of DBS processing method was verified.Judging whether there was cross contamination of DBS punching by the thalassemia gene test results of blank hole which were punched in the blank filter paper between thalassemia positive DBS.The DBS storage stability in thalassemia gene test was verified by detecting the DBS which were dry stored at room temperature for 6 and 9 months.Results 5 circles (3 mm in diameter)DBS were vibrating eluted at 55 ℃ for 1 hour,the DNA concentration extracted from the elution solution was 10-20 ng/μL,which was dissolved in 50 μL solution,and the DNA quality was good.The thalassemia gene test results of DBS and whole blood were the same,and the DBS results of two thalassemia gene test methods were the same too. The cross contamination of DBS punching was not detected in thalassemia gene test.The DBS which were dry stored at room tem-perature for 6 and 9 months could be stably performed thalassemia gene test.Conclusion DBS could be used to perform thalassemia gene test,which is accurate,convenient and stable.It is an ideal way for specimen referral of thalassemia gene test.

Isolation of human genomic DNA from archival dried blood spots for neonatal disease screening and its application to methylation detection / 中华实用儿科临床杂志

Objective To establish an effective DNA isolation method for neonatal disease screening,so as to explore its application to the methylation detection.Methods The 20 dried blood spots samples were randomly divided into 2 groups according to the gender:the traditional method group (n =10) and the improved kit method group(n =10).The DNA quality was evaluated based on its concentration,integrity and whether it could be used in polymerase chain reaction (PCR).These DNA samples with or without bisulfite treatment were used as template in the methylation-specific polymerase chain reaction (MSP).The methylation levels of Leptin and tumor necrosis factor-α (TNF-α) gene promoter region were detected.Results DNA concentration of the improved kit method [(5.70 ± 0.81) mg/L] was significantly higher than that of the traditional method [(3.50 ± 0.45) mg/L] (t =2.79,P ＜ 0.05),and biochemical analyzer analysis showed a better DNA integrity.Agarose gel electrophoresis revealed that 18S gene fragment could be successfully amplified by PCR method,suggesting its potential application to PCR study.MSP results showed different DNA methylation levels of Leptin and TNF-α genes promoter regions from various samples.Conclusions The improved kit method can effectively extract DNA from dried blood spots samples,and these DNA can be used in methylation research.The study can provide a new research direction and technical method to reveal the pathogenesis of disease from the perspective of DNA methylation.

Comparison of Different Extraction Methods of Genomic DNA of Cordia Dicholoma Seeds / 中国中医药信息杂志

Objective To extract high quality genomic DNA of Cordia dicholoma seeds by using different methods;To provide references for researches on genomic DNA of Cordia dicholoma seeds. Methods Genomic DNA of Cordia dicholoma seeds was extracted through improved CTAB method and improved SDS method. Purity and concentration of obtained DNA were detected by spectrophotometry and agarose gel electrophoresis. Results The results of spectrophotometry showed that the purity of genomic DNA obtained through improved CTAB method was better than improved SDS method. Genomic DNA extracted through improved CTAB method was without protein and RNA pollution. The results of agarose gel electrophoresis showed that electrophoresis of genomic DNA obtained through the two methods both had the main belt. However, genomic DNA extracted through improved SDS method degraded more than improved CTAB method. Conclusion Improved CTAB method can obtain relatively high quality genomic DNA of Cordia dicholoma seeds.

An Easy, Rapid, and Cost-Effective Method for DNA Extraction from Various Lichen Taxa and Specimens Suitable for Analysis of Fungal and Algal Strains

Lichen studies, including biodiversity, phylogenetic relationships, and conservation concerns require definitive species identification, however many lichens can be challenging to identify at the species level. Molecular techniques have shown efficacy in discriminating among lichen taxa, however, obtaining genomic DNA from herbarium and fresh lichen thalli by conventional methods has been difficult, because lichens contain high proteins, polysaccharides, and other complex compounds in their cell walls. Here we report a rapid, easy, and inexpensive protocol for extracting PCR-quality DNA from various lichen species. This method involves the following two steps: first, cell breakage using a beadbeater; and second, extraction, isolation, and precipitation of genomic DNA. The procedure requires approximately 10 mg of lichen thalli and can be completed within 20 min. The obtained DNAs were of sufficient quality and quantity to amplify the internal transcribed spacer region from the fungal and algal lichen components, as well as to sequence the amplified products. In addition, 26 different lichen taxa were tested, resulting in successful PCR products. The results of this study validated the experimental protocols, and clearly demonstrated the efficacy and value of our KCl extraction method applied in the fungal and algal samples.

Cloning and analysis of Actin from Paeonia lactiflora / 中草药

Objective: Genomic gene encoding Actin in Paeonia lactiflora was cloned in order to clarify the gene organization and its expression levels in different tissues in herbaceous peony. Methods: Based on cDNA sequences of Actin genes isolated from P. lactiflora reported by our laboratory, one pair of PCR primers was designed. PCR products of Actin genomic gene were successfully amplified with total genomic DNA extracted from herbecous peony cv. "Taohuafeixue" as template by high-fidelity DNA polymerase KOD-Plus, and then they were cloned to pMD18-T vector and be sequenced. The exons and introns of Actin gene were predicted with bioinformatic softwares. The homology was analyzed by Blastn at the nucleotide level and the molecular phylogenetic tree was constructed with MEGA5.0 software. Based on sequencing results, one pair of PCR primers was designed, and the expression levels of Actin gene in the roots, stems, flowers, and leaves in herbaceous peony were semi-quantified. Results: The sequencing results showed that Actin gene of herbaceous peony was 1405 bp length, which contained four exons and three introns. All the splicing sites in three introns were conservative at 5' donor with GU and 3' receptor with AG. It encoded 377 amino acids, and GenBank accession number was KF363830. A pair of PCR primers was designed and one of them was spaned the first intron, which could effectively prevent the false postive by genomic DNA contamination. The semi-quantitative RT-PCR analysis showed that the expression levels of Actin gene in the roots, stems, leaves, and flowers of herbaceous peony were almost constant. Conclusion: It is the first report on cloning and gene organization clarification of genomic gene encoding Actin in P. lactiflora. The semi-quantitative analyses indicate that Actin gene can be used as the internal standard gene for the expression analysis of the functional genes in herbaceous peony.

The application of buccal mucosa swab in analysis of gene polymorphism in children with ASD / 中国神经精神疾病杂志

Objective To investigate the feasibility of buccal mucosa swab method to isolate genomic DNA for au-tism spectrum disorders (ASD)-related genetic screening. Methods Buccal mucosa swabs and blood were collected from 41 children with ASD. Genomic DNA was extracted from either blood by using a commercial genomic DNA kit or buccal mucosa swab by using phenol-chloroform-isoamyl alcohol method. The concentration, total quality and purity of genomic DNA were compared between these two methods. Genotyping of the ASD-related methylenetetra-hydrofolate reductase (MTHFR) gene C677T locus was analyzed using PCR-restriction enzymatic digestion and sanger sequencing was per-formed for validation. Results The total quality [(5.87±2.58)μg vs. (2.00±0.92)μg] and concentration [(143.25±72.78) mg/L vs. (66.68±24.43) mg/L] of genomic DNA extracted from buccal mucosa swab were higher than that form blood (P0.05). Genotyping analysis of MTHFR was also consistent between these two methods. Conclusion Buccal mucosa swab is a simple, non-invasive and reliable meth-od to obtain genomic DNA, which can partially replace blood for analysis of ASD-related gene polymorphisms.

Excised radicle tips as a source of genomic DNA for PCR-based genotyping and melting curve analysis in cotton.

Genomic DNA isolation in cotton is complicated because of the presence of secondary metabolites that are inhibitory to PCR amplification. We report here that radicle tips, but not other parts of cotton seedlings, yield high-quality DNA that is readily amenable for PCR. The radicle-tip-excised seedlings retain viability because of the formation of adventitious roots. We demonstrate the utility of this method in distinguishing homozygotes from heterozygotes in a cotton breeding population and in hybrid seed purity testing.

An improved method for genomic DNA extraction from strawberry leaves / Otimização de um método para extração de DNA genômico a partir de folhas de morangueiro

Several extraction methods of genomic DNA for identification and characterization of genetic diversity in different plant species are routinely applied during molecular analysis. However, the presence of undesirable compounds such as polyphenols and polysaccharides is one of the biggest problems faced during the isolation and purification of high quality DNA in plants. Therefore, achievement of fast and accurate methods for DNA extraction is crucial in order to produce pure samples. Leaves of strawberry genotypes (Fragaria ananassa) have high contents of polysaccharides and polyphenols which increase the sample viscosity and decrease the DNA quality, interfering with the PCR performance. Thereby, in this study we evaluated the quality and amount of genomic DNA extracted from young leaves of strawberry after tissue lyophilization and maceration in presence of polivinilpirrolidone (PVP). The CTAB method was used as reference procedure and it was modified to improve the DNA extraction. The modifications consisted of tissue lyophilization overnight until it was completely freeze-dried and addition of PVP during the tissue maceration in liquid nitrogen. The results showed the efficiency and reliability of the modified method compared to the unmodified method, indicating that combination of lyophilization and PVP improve the quality and amount of the DNA extracted from strawberry leaves.

Vários métodos de extração de DNA genômico para a identificação e caracterização da diversidade genética em diferentes espécies de plantas são rotineiramente aplicados durante a análise molecular. Entretanto, a presença de compostos indesejáveis, tais como polifenóis e polissacarídeos, é um dos maiores problemas que ocorrem durante o isolamento e purificação de DNA de alta qualidade em plantas. Dessa forma, o sucesso no desenvolvimento de métodos de extração de DNA rápidos e acurados é crucial para produzir amostras puras. Folhas de genótipos de morangueiro (Fragaria ananassa) têm elevado conteúdo de polissacarídeos e polifenóis que aumentam a viscosidade da amostra e reduzem a qualidade do DNA, interferindo no desempenho da PCR. Neste estudo, avaliamos a qualidade e a quantidade de DNA genômico extraído de folhas jovens de morangueiro após a liofilização do tecido e a maceração na presença de polivinilpirrolidona (PVP). O método CTAB foi utilizado como procedimento de referência e foi modificado para melhorar a extração do DNA. As modificações consistiram na liofilização do tecido a baixa temperatura até que ele tivesse sido desidratado completamente, associada à adição de PVP durante a maceração do tecido no nitrogênio líquido. Os resultados demonstraram a eficiência e a confiabilidade do método modificado comparado ao método não modificado, indicando que a combinação da liofilização com PVP melhora a qualidade e quantidade do DNA extraído de folhas de morangueiro.