To investigate the genetic diversity among wild Dipsacus asperoides in China, 66 germplasmic resources of D. asperoides were analyzed by Start Codon Targeted Polymorphism (SCoT) molecular markers. Genetic distance was calculated by TREECONW software and the systematic diagram of genetic relationship was clustered by UPGMA method. The results showed that the totals of 181 bands were detected using 20 primers , among which 109 were polymorphic bands. The average percentage of polymorphic bands was 60.13%. Genetic distance changed from 0.030 6 to 0.181 4. The clustering results showed that there was no significant correlation between the classification of the wild D. asperoides and their geographical origin. The relatively high genetic diversity of D. asperoides provides the basis for breeding new varieties.
China ; DNA Primers ; genetics ; DNA, Plant ; genetics ; Dipsacaceae ; chemistry ; classification ; genetics ; Genetic Variation ; Phylogeny ; Polymorphism, Genetic

OBJECTIVETo make the kit with witch to identify Penis et Testis Cervi with molecular taxonomy.

METHODThe mtDNA of sika and red deer from different areas was amplified by PCR and sequenced. Compared with the mtDNA of bovine and horse from witch the false medicines were made, characteristic segments of deer were found. We selected one as the species distinctive PCR primer of deer.

RESULTThe kit made up with this primer and related reagents could be used to discern Penis et Testis Cervi from the false medicine.

CONCLUSIONIt is a scientific, steady, accurate and convenient way to identify Penis et Testis Cervi with molecular taxonomy.

Animals ; Cattle ; genetics ; DNA ; genetics ; DNA Primers ; DNA, Mitochondrial ; genetics ; Deer ; classification ; genetics ; Drug Contamination ; Horses ; genetics ; Male ; Materia Medica ; chemistry ; Penis ; chemistry ; Testis ; chemistry

OBJECTIVETo determine whether 3'phosphorothioate-modified-2 terminal mismatched primers can turn off DNA polymerization mediated by Exo(+) polymerase.

METHODSTwo-directional primer extension was performed using polymerase with and without 3' exonuclease activity. The effects of unmodified primers and 3' phosphorothioate-modified primers on primer extension were evaluated.

RESULTSExo(-) polymerase yielded products from matched and mismatched primers regardless of their modification. However, 3' phosphorothioate-modified primers with a single base mismatch at -2 position worked similarly to the terminal (-1) mismatched primers in triggering the novelly reported "off-switch" of Exo(+) polymerase.

CONCLUSIONThese data suggested that the "off-switch" can be of enormous application in the diagnosis of single gene diseases and in the association studies by single nucleotide polymorphism screening.

DNA Primers ; chemistry ; genetics ; Exonucleases ; metabolism ; Humans ; Phosphorothioate Oligonucleotides ; chemistry ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide

The purpose of this study was to develop pneumococcal typing by multiplex PCR and compare it with conventional serotyping by quellung reaction. Pneumococcal strains used in this study included 77 isolates from clinical specimens collected from children at Seoul National University Children's Hospital from 2006 to 2010. These strains were selected as they represented 26 different serotypes previously determined by quellung reaction. Molecular type was determined by 8 sequential multiplex PCR assays. Bacterial DNA extracted from cultured colonies was used as a template for PCR, and primers used in this study were based on cps operon sequences. Types 6A, 6B, 6C, and 6D were assigned based on the presence of wciNbeta and/or wciP genes in 2 simplex PCRs and sequencing. All 77 isolates were successfully typed by multiplex PCR assays. Determined types were as follows: 1, 3, 4, 5, 6A, 6B, 6C, 6D, 7C, 7F, 9V, 10A, 11A, 12F, 13, 14, 15A, 15B/15C, 19A, 19F, 20, 22F, 23A, 23F, 34, 35B, and 37. The results according to the PCR assays were in complete concordance with those determined by conventional quellung reaction. The multiplex PCR assay is highly reliable and potentially reduces reliance upon conventional serotyping.
Child ; DNA Primers/chemistry/metabolism ; DNA, Bacterial/chemistry/genetics ; Humans ; Multiplex Polymerase Chain Reaction ; Pneumococcal Infections/microbiology ; Serotyping ; Streptococcus pneumoniae/*classification/genetics/isolation & purification

High resolution melting (HRM) , an important technology for genotyping and mutation scanning, has broad prospects in the authenticity of traditional Chinese medicine. This paper selected universal CO I primers and used HRM to establish a new method for authenticity of Hairy Antler. PCR was conducted at the annealing temperature of 60 °C and 45 cycles. The range of the DNA template concentration, the primer concentration and the Mg2+ ion concentration were further optimized. The results showed that the Tm values of Cervus nippon were (81.96 ± 0.07), (84.51 ± 0.03) °C and Cervus elaphus was(82.58 ± 0.13), (85.95 ± 0.05) °C with 10-100 mg · L(-1) DNA template, 0.2 µLmol · L(-1) primer, 2.0 mmol · L(-1) Mg2+. This method can authenticate of hairy antler and is simple, fast, high-throughput, visualization.
Animals ; Antlers ; chemistry ; DNA ; chemistry ; genetics ; DNA Primers ; genetics ; Deer ; classification ; genetics ; Genotype ; Medicine, Chinese Traditional ; standards ; Polymerase Chain Reaction ; Transition Temperature

Outwardly rectifying swelling-activated chloride conductance (ICl,Swell) in rabbit heart plays a critical role in cardioprotection following ischemic preconditioning (IP). But the functional characterization and molecular basis of this chloride conductance in rabbit heart ventricular myocytes is not clear. Candidate chloride channel clones (e.g. ClC-2, ClC-3, ClC-4 and ClC-5) were determined using RT-PCR and Western blot analysis. Whole cell ICl,Swell was recorded from isolated rabbit ventricular myocytes using patch clamp techniques during hypo-osmotic stress. The inhibitory effects of 4,4' isothiocyanato-2,2-disulfonic acid (DIDS), 5-nitro-2(3-phenylroylamino) benzoic acid (NPPB) and indanyloxyacetic acid 94 (IAA-94) on ICl,Swell were examined. The expected size of PCR products for ClC-2, ClC-3 and ClC-4 but not for ClC-5 was obtained. ClC-2 and ClC-3 expression was confirmed by automated fluorescent DNA sequencing. RT-PCR and Western blot showed that ClC-4 was expressed in abundance and ClC-2 was expressed at somewhat lower levels. The biological and pharmacological properties of I(Cl,Swell), including outward rectification, activation due to cell volume change, sensitivity to DIDS, IAA-94 and NPPB were identical to those known properties of ICl,Swell in exogenously expressed systems and other mammals hearts. It was concluded that ClC-3 or ClC-4 might be responsible for the outwardly rectifying part of ICl,Swell and may be the molecular targets of cardioprotection associated with ischemic preconditioning or hypo-osmotic shock.
Biophysics/methods ; Chlorides/*chemistry ; Chlorides/metabolism ; DNA Primers/chemistry ; Electrophysiology/methods ; Gene Expression Regulation ; Glycolates/pharmacology ; Heart Ventricles/*cytology ; Ischemic Preconditioning ; Muscle Cells/*cytology ; Osmosis ; Sequence Analysis, DNA

The different growing habitats of Bupleurum chinense were investigated in Donglin mountain & Wuling mountain areas, the saikosaponin a and d in samples of B. chinense collected from different habitats were determined by HPLC. Results showed that B. chinense distributed in various habitats, such as meadow, understory and brushy. Significant differences of saikosaponin contents were observed. The higher saikosaponins contents were showed in samples from meadow habitats, while the lower saikosaponins contents in samples from understory and brushy habitats. The ventilation situation and light condition showed positive correlation with the saikosaponins accumulation in B. chinense. It could be concluded that growing habitats play an important role in accumulation of saikosaponins in B. chinense.
Bupleurum ; chemistry ; China ; Chromatography, High Pressure Liquid ; DNA Primers ; genetics ; Ecosystem ; Oleanolic Acid ; analogs & derivatives ; analysis ; Saponins ; analysis

OBJECTIVETo establish a convenient, quick and accurate molecular method for the identification of crude drugs of antlers due to the difficult discrimination between the genuine antler and its adulterants.

METHODAccording to the alignment analysis of full length sequences of Cyth gene from closely relate species of Cervus, one pair of allele-specific diagnostic PCR primers was designed. Factors such as annealing temperature, dosage of polymerase, times of cycles and dosage of template DNA that influence the PCR results were also investigated.

RESULTBased on the study mentioned above, about 323 bp positive band was amplified under the annealing temperature of 65 degrees C in the total volume of 25 microL PCR reaction using the genuine antler DNA as the template. Sequencing results proved that the positive band was the fragment of Cytb gene from both C. elaphus Linnaeus and C. nippon Temminck.

CONCLUSIONThe established method, with higher specificity and reproducibility, could accurately differentiate genuine antler from its adulterants and would be widely used in Cervus related Chinese crude drugs' identification.

Alleles ; Animals ; Antlers ; chemistry ; China ; DNA Primers ; genetics ; Deer ; genetics ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; Species Specificity

In this paper, Liuwei Dihuang pill was used to study the identification of Chinese patent medicine by fluorescence sequencing typing technology. The DNA of Paeonia suffruticosa was used as template to amplify by five pair of FAM fluorescence labeling primers. Then, the amplified products were sequenced. The sequencing results were analyzed by GeneMarker V1.80 to screen the best fluorescence labeling primers. As a result, psbA-trnH fluorescence labeling primer was used to identify the raw materials of Liuwei Dihuang pill. The results showed that three kinds of raw plant medicinal materials in Liuwei Dihuang pill were able to be correctly identified by psbA-trnH fluorescence labeling primer. The fluorescence sequencing typing technology can stably and accurately distinguish raw medicinal materials in Chinese patent medicine.
DNA Primers ; chemistry ; genetics ; DNA, Plant ; chemistry ; genetics ; Drugs, Chinese Herbal ; chemistry ; standards ; Fluorescent Dyes ; chemistry ; Plants, Medicinal ; chemistry ; genetics ; Polymerase Chain Reaction ; instrumentation ; methods ; Quality Control ; Staining and Labeling

OBJECTIVETo develop a high-throughput screening assay for Farnesoid X receptor (FXR) agonists based on mammalian one-hybrid system (a chimera receptor gene system) for the purpose of identifying new lead compounds for dyslipidaemia drug from the chemical library.

METHODScDNA encoding the human FXR ligand binding domain (LBD) was amplified by RT-PCR from a human liver total mRNA and fused to the DNA binding domain (DBD) of yeast GAL4 of pBIND to construct a GAL4-FXR (LBD) chimera expression plasmid. Five copies of the GAL4 DNA binding site were synthesized and inserted into upstream of the SV40 promoter of pGL3-promoter vector to construct a reporter plasmid pG5-SV40 Luc. The assay was developed by transient co-transfection with pG5-SV40 Luc reporter plasmid and pBIND-FXR-LBD (189-472) chimera expression plasmid.

RESULTSAfter optimization, CDCA, a FXR natural agonist, could induce expression of the luciferase gene in a dose-dependent manner, and had a signal/noise ratio of 10 and Z' factor value of 0.65.

CONCLUSIONA stable and sensitive cell-based high-throughput screening model can be used in high-throughput screening for FXR agonists from the synthetic and natural compound library.

Base Sequence ; Cell Line ; DNA Primers ; DNA, Complementary ; DNA-Binding Proteins ; agonists ; chemistry ; genetics ; Humans ; Hypolipidemic Agents ; analysis ; Plasmids ; Receptors, Cytoplasmic and Nuclear ; agonists ; chemistry ; genetics ; Reproducibility of Results ; Transcription Factors ; agonists ; chemistry ; genetics ; Transfection