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

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

OBJECTIVETo establish an exonuclease protection mediated polymerase chain reaction (PCR) assay for the non-radioactive, sensitive detection of the binding of protein and DNA.

METHODSThe 1 pmol/L-10 nmol/L 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) dissolved in dimethyl sulphoxide (DMSO), was added into 100 microl SD rat hepatic cytosol in vitro, which contained different amount of aromatic hydrocarbon receptors (AhR) and relative proteins, and ligand-AhR-DRE complex were formed in addition to 1 fmol/L-100 nmol/L DNAs probes containing the sequence of DRE. With the digestion of Exonuclease III and S1 nuclease, free DNAs were digested to oligonucleotide and binding DNA remained due to protein (AhR) protection and be amplified by PCR. The results of PCRs were shown by loading on 2% agarose electrophoresis. DMSO was used as negative control and blank control was set up.

RESULTSTarget DNA (285 bp) could be observed in the ligand groups, but not in the control group. The minimal amount of receptor was 2.5 fmol/L and the minimal amount of DNA probes was 2 fmol.

CONCLUSIONSExonuclease protection mediated PCR assay should be a good non-radioactive tool to quantify the interaction of protein and DNA with high sensitivity and simplicity.

Animals ; Aryl Hydrocarbon Receptor Nuclear Translocator ; genetics ; metabolism ; Binding, Competitive ; DNA Probes ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Exonucleases ; metabolism ; Male ; Polymerase Chain Reaction ; methods ; Rats ; Rats, Sprague-Dawley ; Receptors, Aryl Hydrocarbon ; genetics ; metabolism

A strain, MMR003, used for D-p-HPG production in industry was classified as Burkholderia pickettii by morphological observation and biochemical characterization. The gene encoding the D-hydantoinase enzyme was cloned, sequenced and expressed in Escherichia coli. The nucleotide sequence of the 5.0 kb insert of subclone pXZ-total was determined. One open reading frame of 1374 bp was found and predicted to encode a polypeptide consisting of 458 amino acids in size of 50 kD. The amino acid sequence alignment of D-hydantoinase from Burkholderia pickettii shows the 85% homologous with the corresponding enzyme from Agrobacterium radiobacter NRRL B11291. The D-hydantoinase gene (dha) harboured in the plasmid pXZPH2 in E. coli BL21(DE3) was highly expressed by IPTG induction. The D-hydantoinase activity for D, L-p-hydroxyphenylhydantion is 0.66 u/mL broth, which is 2-fold increase compared to the parent strain Burkholderia pickettii.
Amidohydrolases ; genetics ; metabolism ; Amino Acid Sequence ; Animals ; Burkholderia ; classification ; enzymology ; genetics ; Cloning, Molecular ; DNA Probes ; Escherichia coli ; Gene Expression ; Molecular Sequence Data ; Recombination, Genetic ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid

OBJECTIVETo establish a sensitive and specific technique for detecting HBV DNA in serum using HBV DNA probe labeled directly by alkaline phosphatase (AlkPhos Direc probe).

METHODSThe probe that purified HBV DNA sequence was labeled directly by alkaline phosphatase and chemiluminescent substrate CDP-star for AP was used in the hybridization assay. HBV DNA was detected by autoradiography on the film. The test compared the chemiluminescen dot blot hybridization assay for 80 samples with digoxigenin-labeled HBV DNA probe detective method. The correlation of 70 samples test results between fluorescent quantitative HBV DNA PCR method and dot blot hybridization assay by AlkPhos Direc probe was analysed.

RESULTSThe sensitivity of the probe labeled directly by alkaline phosphatase was 10pg at least. The coincidence was 100% compared with digoxigenin-labeled HBV DNA probe detection. A correlation coefficient of HBV DNA quantitative results between fluorescent quantitative HBV DNA PCR (QPCR) method and dot blot hybridization assay by AlkPhos Direc probe was 0.98 (P<0.01).

CONCLUSIONSThe method detecting HBV DNA in serum by HBV DNA AlkPhos Direc probe is sensitive and specific. The results between two methods with AlkPhos Direc and digoxigenin-labeled HBV DNA probe are coincident completely. The correlation of HBV DNA quantitative results between fluorescent QPCR method and dot blot hybridization assay by AlkPhos Direc probe is satisfactory.

Alkaline Phosphatase ; chemistry ; metabolism ; Animals ; DNA Probes ; chemistry ; genetics ; DNA, Viral ; blood ; genetics ; Hepatitis B ; blood ; diagnosis ; virology ; Hepatitis B virus ; genetics ; Humans ; Molecular Diagnostic Techniques ; methods ; standards ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

The aim of this study was to develop and validate an oligonucleotide suspension array for rapid identification of 15 bacterial species responsible for bacteremia, particularly prevalent in Chinese hospitals. The multiplexed array, based on the QIAGEN LiquiChip Workstation, included 15 oligonucleotide probes which were covalently bound to different bead sets. PCR amplicons of a variable region of the bacterial 23S rRNA genes were hybridized to the bead-bound probes. Thirty-eight strains belonging to 15 species were correctly identified on the basis of their corresponding species-specific hybridization profiles. The results show that the suspension array, in a single assay, can differentiate isolates over a wide range of strains and species, and suggest the potential utility of suspension array system to clinical laboratory diagnosis.
Bacteremia ; diagnosis ; genetics ; Bacterial Typing Techniques ; Bacteriological Techniques ; DNA Probes ; Genetic Techniques ; Listeria monocytogenes ; metabolism ; Nucleic Acid Hybridization ; Oligonucleotide Array Sequence Analysis ; Oligonucleotides ; chemistry ; RNA, Ribosomal ; chemistry ; RNA, Ribosomal, 23S ; genetics ; Stem Cells

OBJECTIVETo establish a high sensitive and specific method of interphase fluorescence in situ hybridization (IFISH) to detect the low-frequency human cells in human/goat xenogeneic models.

METHODSHuman-specific Y-chromosome satellite DNA CEPY and 17-chromosome satellite DNA p17H8 were used as probes for IFISH. The peripheral blood samples from 2 goats transplanted with human male hematopoietic stem cells (HSC), 1 normal negative goat and 1 normal man were analyzed. The actual FISH efficiency was confirmed by serial dilutions (1/100, 1/500 and 1/1000) of the cell mixture of normal man and normal negative goat. A set of signal scoring criteria was determined to guarantee the stability and reliability of the method.

RESULTSPositive cell (human cell) frequencies were consistent with the established frequencies for the human/goat cell mixture. The average frequencies of positive cells were 98.60% (CEPY) and 100% (p17H8) for normal man, 0 for normal negative goat, 0.23% (CEPY) and 0.11% (p17H8) for human/goat xenogeneic models. The results demonstrated that low-frequency human cells (male cells confirmed by Y-chromosome probe) existed in human/goat xenogeneic models.

CONCLUSIONThe IFISH developed in this study is of high sensitivity and specificity and can identify the actual frequency of human cells, which offers a direct, sensitive and specific approach to the detection of low-frequency human cells in human/goat xenogeneic models.

Animals ; Chromosomes, Human, Pair 17 ; genetics ; Chromosomes, Human, Y ; genetics ; DNA Probes ; Goats ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; cytology ; metabolism ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Interphase ; genetics ; Microsatellite Repeats ; genetics ; Transplantation Chimera ; blood ; genetics ; Transplantation, Heterologous

BACKGROUND: A peptide nucleic acid (PNA) probe-based real-time PCR (PNAqPCR(TM) TB/NTM detection kit; PANAGENE, Korea) assay has been recently developed for the simultaneous detection of Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM) in clinical specimens. The study was aimed at evaluation of the performance of PNA probe-based real-time PCR in respiratory specimens. METHODS: To evaluate potential cross-reactivity, the extracted DNA specimens from Mycobacterium species and non-mycobacterial species were tested using PNA probe-based real-time PCR assay. A total of 531 respiratory specimens (482 sputum specimens and 49 bronchoalveolar washing fluid specimens) were collected from 230 patients in July and August, 2011. All specimens were analyzed for the detection of mycobacteria by direct smear examination, mycobacterial culture, and PNA probe-based real-time PCR assay. RESULTS: In cross-reactivity tests, no false-positive or false-negative results were evident. When the culture method was used as the gold standard test for comparison, PNA probe-based real-time PCR assay for detection of MTBC had a sensitivity and specificity of 96.7% (58/60) and 99.6% (469/471), respectively. Assuming the combination of culture and clinical diagnosis as the standard, the sensitivity and specificity of the new real-time PCR assay for detection of MTBC were 90.6% (58/64) and 99.6% (465/467), respectively. The new real-time PCR for the detection of NTM had a sensitivity and specificity of 69.0% (29/42) and 100% (489/489), respectively. CONCLUSIONS: The new real-time PCR assay may be useful for the detection of MTBC in respiratory specimens and for discrimination of NTM from MTBC.
Bronchoalveolar Lavage Fluid/microbiology ; DNA Probes/chemistry/metabolism ; DNA, Bacterial/*analysis ; Humans ; Molecular Typing/*methods ; Mycobacterium tuberculosis/*genetics/isolation & purification ; Nontuberculous Mycobacteria/*genetics/isolation & purification ; Nucleic Acid Hybridization ; Peptide Nucleic Acids/chemistry/*metabolism ; *Real-Time Polymerase Chain Reaction ; Respiratory System/*microbiology ; Sputum/microbiology

BACKGROUND: Intrachromosomal amplification of chromosome 21 (iAMP21) is known to be associated with poor prognosis in B-cell ALL (B-ALL). To determine the frequency and clinical characteristics of iAMP21 in Korean B-ALL patients, we performed FISH and multiplex ligation-dependent probe amplification (MLPA) analyses. METHODS: A total of 102 childhood B-ALL patients were screened with ETV6-RUNX1 FISH probes (Abbott Molecular, USA). The presence of an iAMP21 was confirmed by using MLPA P327 iAMP21-ERG probemix (MRC Holland, The Netherlands). RESULTS: iAMP21 was detected in one of the screened B-ALL patients (1/102 patients, 1.0%) who presented the ALL immunophenotype and complex karyotype at initial diagnosis. The patient relapsed twice after bone marrow transplantation. MLPA showed 12.5-Mb and 4.28-Mb regions of amplification and deletion, respectively. CONCLUSIONS: The frequency of iAMP21 is considerable in Korean pediatric patients. Our report suggests that iAMP21 in childhood B-ALL has very unfavorable impact on patient's prognosis. Additional methods such as MLPA analysis is essential to rule out patients with equivocal interphase FISH results.
Adolescent ; Asian Continental Ancestry Group/*genetics ; B-Lymphocytes/*metabolism ; Child ; Child, Preschool ; *Chromosomes, Human, Pair 21 ; Core Binding Factor Alpha 2 Subunit/genetics ; DNA Probes/metabolism ; Female ; Humans ; Immunophenotyping ; In Situ Hybridization, Fluorescence ; Infant ; Infant, Newborn ; Male ; Multiplex Polymerase Chain Reaction ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/*diagnosis/genetics ; Proto-Oncogene Proteins c-ets/genetics ; Repressor Proteins/genetics ; Republic of Korea ; Translocation, Genetic ; Young Adult

The present paper is aimed to detect superparamagnetic iron oxide labeled c-erbB2 oncogene antisense oligonucleotide probe (magnetic antisense probe) connected with SK-Br-3 oncocyte mRNA nucleotide by high resolution atomic force microscope (AFM). We transfected SK-Br-3 oncocyte with magnetic antisense probe, then observed the cells by AFM with high resolution and detected protein expression and magnetic resonance imagine (MRI). The high resolution AFM clearly showed the connection of the oligonucleotide remote end of magnetic antisense probe with the mRNA nucleotide of oncocyte. The expression of e-erbB2 protein in SK-Br3 cells were highly inhibited by using magnetic antisense probe. We then obtained the lowest signal to noise ratio (SNR) of SK-Br-3 oncocyte transfected with magnetic antisense probe by MRI (P<0.05). These experiments demonstrated that the high resolution AFM could be used to show the binding of magnetic antisense probe and SK-Br-3 mRNA of tumor cell nuclear.
Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; DNA, Antisense ; chemistry ; genetics ; Female ; Ferric Compounds ; chemistry ; Genes, erbB-2 ; genetics ; Humans ; Magnetics ; Microscopy, Atomic Force ; methods ; Molecular Probe Techniques ; Nucleic Acid Probes ; chemistry ; genetics ; Oligodeoxyribonucleotides ; chemistry ; genetics ; Oxyphil Cells ; ultrastructure ; RNA, Messenger ; genetics ; metabolism