OBJECTIVETo study the expression pattern of cd99l2 gene during zebrafish development, the RNA probes for whole-mount in situ hybridization were prepared in this study.

METHODSThe cd99l2 fragment obtained by RT-PCR was cloned into pGM-T Easy, then the plasmids were linearized with the restriction enzymes SacII or SalI. Using Sp6 or T(7) RNA polymerase, the digoxingenin-labeled antisense and sense probes were synthesized and confirmed by whole-mount in situ hybridization.

RESULTSThe plasmid cd99l2/pGM-T was constructed. cd99l2 gene expression pattern during embryogenesis of zebrafish was examined using the antisense probe, and intense expression was detected in the central nervous system during zebrafish development.

CONCLUSIONThe antisense probe can be used for study of the spatial and temporal distribution of cd99l2 during zebrafish development using the sense probe as control.

Animals ; Central Nervous System ; embryology ; Cloning, Molecular ; Digoxigenin ; chemistry ; Gene Expression Regulation, Developmental ; Oligonucleotide Probes ; RNA Probes ; Uridine Triphosphate ; chemistry ; Zebrafish ; embryology ; genetics ; Zebrafish Proteins ; genetics

To investigate the prevalence of point mutation in the pre-core (pre-C) region of hepatitis B virus (HBV) DNA, we performed dot blot hybridization and sequencing of enzymatically amplified HBV DNA from the sera of 25 patients with HBeAg-positive and 32 patients with HBeAg-negative chronic liver diseases. The pre-C region of HBV DNA was successfully amplified by polymerase chain reaction (PCR) from 55 (96.5%) of 57 sera. According to the status of serum HBeAg, HBV DNA was amplified from all 25 sera of HBeAg-positive patients and 30 (93.8%) of 32 sera of HBeAg-negative patients. All amplified DNA from the sera of 25 patients with HBeAg-positive and that from 28 (93.3%) of 30 patients with HBeAg-negative chronic liver diseases hybridized with the wild type probe. In addition, that from 5 (20.0%) among 25 patients with HBeAg-positive and 16 (53.3%) among 30 patients with HBeAg-negative chronic liver diseases hybridized also with the mutant type probe. These results suggest that the prevalence of point mutation in the pre-C region of HBV DNA is relatively high in patients with HBeAg-negative chronic liver diseases and further study is mandatory to identify the significance of this mutation.
Base Sequence ; Chronic Disease ; DNA, Viral/*genetics ; Hepatitis B Virus/*genetics ; Human ; Liver Diseases/*genetics ; Molecular Probes/genetics ; Molecular Sequence Data ; *Mutation

Two oligonucleotide probes are permitted to anneal to the nucleic acid target of interest so that the ends of two probes immediately become adjacent to each other. The ligase can then efficiently join the two juxtaposed oligonucleotide probes by the formation of a phosphodiester bond if and only if perfectly matched base-pairs at the nick are present. During past 20 years, many ligase-mediated techniques have been developed for analyzing various bio-molecules, such as known/unknown point mutations, small-scale insertions and deletions, CpG islands methylation, large sets of single nucleotide polymorphisms (SNPs), specific proteins and DNA regions with which some other proteins can interact. Since the ligation reaction can be easily integrated into other techniques, certain advances have been already achieved. These novel approaches retain high accuracy through multiple hybridization and enzymatic processing events, and provide inherent quality control checking. In this article, we provide a comprehensive review of the ligase-mediated techniques for bio-molecular analysis.
CpG Islands ; genetics ; DNA Methylation ; Ligases ; metabolism ; Molecular Probe Techniques ; Mutation ; Oligonucleotide Probes ; biosynthesis ; chemical synthesis ; genetics ; Polymorphism, Single Nucleotide

Nuclear oncolgy is important in the diagnosis, staging, and long-term surveillance of a number of cancers. Over the past 10 years there has been an explosion of new radioisotopic tracers aimed at detecting, staging and eventually treating tumors. Clinicians and oncologists can now use specific radiolabeled metabolic tracers, monoclonal antibodies, and molecular probes based on the sequencing of the human genome. The current applications of positron emission tomography (PET) in oncology have included characterizing tumor lesions, differentiating recurrent disease from treatment effects, staging tumors, evaluating the extent of disease, and monitoring therapy. The future developments in medicine may use the unique capabilities of PET not only in diagnostic imaging but also in molecular medicine and genetics. Radioimmunoscintigraphy is a technique which uses radiolabeled antibodies to visualize tumors, taking advantage of antigens preferentially expressed by malignant tissue. However, the implementation of radiolabeled antibodies as "magic bullets" for detection and treatment of diseases such as cancer has required addressing several shortcomings of murine monoclonal antibodies. Genetic engineering provides a powerful approach for redesigning antibodies for use in oncologic applications in vivo. Recently, noninvasive molecular imaging has been developed. Most current molecular imaging strategies are "indirect" and involve the coupling of a "reporter gene" with a complementary "reporter probe". Imaging the level of probe accumulation provides indirect information related to the level of reporter gene expression. In this article, the author discuss the current status of PET, radioimmunoscintigraphy, gene imaging and receptor imaging with a brief review on nuclear oncology.
Antibodies ; Antibodies, Monoclonal ; Diagnosis ; Diagnostic Imaging ; Explosions ; Genes, Reporter ; Genetic Engineering ; Genetics ; Genome, Human ; Humans ; Molecular Imaging ; Molecular Medicine ; Molecular Probes ; Nuclear Medicine* ; Positron-Emission Tomography ; Radioimmunodetection

OBJECTIVETo develop a TaqMan real-time PCR for the detection of Aeromonas hydrophila.

METHODSThe conserved region of major adhesion gene of Aeromonas hydrophila (aha) was used to design primers and TaqMan probe. A total of six concentration gradients for forward and reverse primers ranging from 200 -700 nmol/L were chosen, and four concentration gradients for probe ranging from 100 - 400 nmol/L were chosen. And then the concentration of primers and probe were optimized by ANOVA of completely randomized design respectively. The specificity of the established method was evaluated by using bacteria as contrast, including 45 strains Vibrio cholerae, 20 strains Vibrio parahaemolyticus, 10 strains Vibrio fluvialis, 4 strains Vibrio mimicus, 5 strains Vibrio vulnificus, 1 strain Vibrio alginolyticus, 1 strain Vibrio furnissii, 5 strains Salmonella, 10 strains Shigella and 2 strains Plesiomonas shigelloides. The sensitivity, bacterial sensitivity and DNA sensitivity included,were evaluated. The stool of healthy people was contaminated by Aeromonas hydrophila artificially, and the ability of the established TaqMan real-time PCR system for detection of Aeromonas hydrophila was also evaluated.

RESULTSThe cycle threshold (Ct) value deserved from 6 groups of primers concentration gradient was (x +/- s):20.69 +/- 0.33, 20.72 +/- 0.21, 20.81 +/- 0.12, 20.74 +/- 0.12, 20.51 +/- 0.16 and 20.69 +/- 0.11, respectively, and the concentration of forward primer and reverse primer was determined to be 200 nmol/L (F = 1.33, P = 0.28). The Ct value deserved from 4 groups of probe concentration gradient was (x +/- s):20.56 +/- 0.08, 20.82 +/- 0.05, 20.82 +/- 0.11 and 20.93 +/- 0.09, respectively, and the concentration of probe was determined to be 100 nmol/L (F = 5.26, P = 0.01). The bacterial sensitivity and DNA sensitivity were 80 CFU/ml and 100 fg/microl respectively, and the sensitivity to detect Aeromonas hydrophila from stool was 8 x 10(3) CFU/ml, which might be 8 CFU/ml after 8 hours' enrichment. No amplification was observed in the templates of other bacterial.

CONCLUSIONThe TaqMan real-time PCR method targeting the aha gene of Aeromonas hydrophila had a high sensitivity and specificity and might be used to detect Aeromonas hydrophila from pure bacterial and stool rapidly.

Aeromonas hydrophila ; genetics ; DNA Primers ; Genes, Bacterial ; Molecular Probes ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Species Specificity

A quantitative real-time PCR assay was developed to measure the proviral load of human T-lymphotropic virus type I (HTLV-I) in peripheral blood. The technology utilizes special primers and Taqman MGB fluorescence probe to measure amplification products from the gag-pro-pol polyprotein gene of HTLV-I. HTLV-I copy number was normalized to the amount of cellular DNA by quantitation of the beta-actin gene, The amplification system was sensitive to detect 5 copy/microL. The standard curve had a good linearity when the quantity for the gene was between 10(3) and 10(7) copy/microL (R2 = 0.999). Good reproducibility was observed in each intra- and inter-assay. We also measured proviral load in peripheral blood in 12 HTLV-I seropositive former blood donors. Proviral load for HTLV-I infected donors ranged from 0.015 to 12.819 copy/cell in WBC with the mean of 3.116 copy/cell.
Gene Products, gag ; genetics ; Gene Products, pol ; genetics ; Human T-lymphotropic virus 1 ; genetics ; isolation & purification ; Humans ; Molecular Probes ; Polymerase Chain Reaction ; methods ; Viral Proteins ; genetics

We have investigated the use of a nested polymerase chain reaction(PCR) assay with Y-specific sequence from the DYS 14 locus on the short arm of Y-chromosome for prenatal sex determination in the peripheral blood of 22 pregnant women who participated in the antenatal genetic diagnosis program. The sensitivity and specificity of the nested PCR using DYS 14 locus primers(Y1.5,Y1.6, and Y1.7,Y1.8) were 76.4% and 55.5%, respectively. In terms of gestational age, positive predictive values of 66.6%, 66.6%, and 80% were obtained for the first, second, and third trimester respectively. The corresponding negative predictive values were 50%, 50%, and 100% respectively. Male specific band was positive in three of the six cases of female bearing women and male specific band was negative in three of the seven cases of male bearing women during 9-16 gestational weeks showing low sensitivity. But all cases except one show the male specific band during the male fetus and all female fetuses did not show the male specific 198 base pair band during 18 approximately 40 gestational weeks. This study suggests that prenatal sex determination by PCR employing maternal peripheral blood was usually possible in late pregnancy but less reliable in early pregnancy. It seems that if we used a method separating fetal cells from maternal blood and then run PCR on these cells with DYS 14 locus primers we could make a fairly accurate fetal sex determination.
Base Sequence ; Female ; Fetus/*physiology ; Gestational Age ; Human ; Male ; Molecular Probes/genetics ; Molecular Sequence Data ; *Polymerase Chain Reaction ; Pregnancy/*blood ; *Sex Determination (Analysis)

DNA stable-isotope probing (DNA-SIP) is a recently developed method with which the incorporation of stable isotope from a labeled substrate is used to identify the function of microorganisms in the environment. The technique has now been used in conjunction with metagenomics to establish links between microbial identity and particular metabolic functions. The combination of DNA-SIP and metagenomics not only permits the detection of rare low-abundance species from metagenomic libraries but also facilitates the detection of novel enzymes and bioactive compounds. We summarize recent progress in SIP-metagenomic techniques and applications and discuss prospects for this combined approach in environmental microbiology and biotechnology.
Animals ; DNA ; genetics ; DNA Probes ; chemistry ; genetics ; metabolism ; DNA, Bacterial ; chemistry ; genetics ; metabolism ; Humans ; Isotope Labeling ; methods ; Metagenomics ; methods ; Molecular Probe Techniques ; Sequence Analysis, DNA ; methods

OBJECTIVE: To construct the genomic library of Raji cells and screen it by EBV DNA probe. METHODS: High molecular weight genomic DNA of Raji cells was digested by restriction enzyme BamHI. DNA fragments ranging from 9 to 23 kb were recovered by agarose gel electrophoresis, which were ligated with Lambda DASH II vector BamHI arms pre-treated with calf intestine alkaline phosphatase (CIAP). Ligated DNA was packed in vitro using Gigapack III gold packaging extract. The library was plated on XL1-blue MRA (P2) host strain.Titering and screening of the Raji genomic library were performed. RESULTS: The primary titer of the Raji genomic library was 1.8 x 10(5) pfu/mL, while that of the amplified library was 2.8 x 10(8) pfu/mL. Plaques (1 x 10(5)) were screened with (32)P-labeled EBV DNA probe(EBV genome 5-3271), 4 positive clones were obtained, and 1 of the 4 positive clones was picked out randomly for the second round of plaque screening. All the phage plaques were positive. DNA of the positive clone was extracted and was digested with BamHI. The length of the inserted fragment was 8.5 kb. Sequencing and BLAST analysis revealed that the inserted fragments consisted of the BamHI-W fragment at one end and clone RP11-665A22 on chromosome 15 at the other end. CONCLUSION The successfully established genomic library of Raji cells will provide a basis for cloning the sequences of the EBV junction sites and interpreting the mechanism of oncogenesis of EBV integration.
Base Sequence ; Burkitt Lymphoma ; genetics ; virology ; Chromosomes, Human, Pair 15 ; genetics ; Cloning, Molecular ; DNA Probes ; genetics ; DNA, Viral ; genetics ; Gene Expression Profiling ; Genes, Neoplasm ; genetics ; Genomic Library ; Herpesvirus 4, Human ; genetics ; Humans ; Molecular Sequence Data ; Tumor Cells, Cultured

OBJECTIVETo prepare oligo microarrays for hepatitis virus detection and genotyping.

METHODSBy analyzing the DNA or cDNA of HBV, HDV and 4 different genotypes of HCV with the BLAST program, a group of specific sequences for the candidate probes was specified. Array Designer 3.0 software was applied to analyze the candidates to select probes with high specificity, identical length and similar melting temperature (Tm). Altogether 16, 8 and 68 oligonucleotide probes were designed for diagnosis of HBV, HDV, and genotyping HCV. Following the synthesizing and purification, oligo probes were deposited on oligonucleotide chips as microarrays for hepatitis virus detection and genotyping. The samples were labeled by RD-PCR method. Hybridization results were analyzed to cross out those probes with low specificity and sensitivity, and those with signal to noise ratios (SNR) less than 4.0.

RESULTSTwo types of gene chips were successfully developed: microarrays for HBV and HDV simultaneous detection and for HCV genotyping.

CONCLUSIONUsing oligo probes to construct gene chips for clinical diagnosis of hepatitis virus is a simple and effective method. It may be widely used in detecting hepatitis viruses and their genotyping in clinical settings.

Base Sequence ; DNA Fingerprinting ; DNA, Viral ; genetics ; Genotype ; Hepatitis B virus ; classification ; genetics ; Molecular Sequence Data ; Oligonucleotide Array Sequence Analysis ; Oligonucleotide Probes ; Sensitivity and Specificity