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PubMed2005

Phylogenetic relationships of honey bees (Hymenoptera:Apinae:Apini) inferred from nuclear and mitochondrial DNA sequence data.

Two different genomic regions (ND2 mitochondrial gene and EF1-alpha intron) were PCR amplified, cloned and sequenced for the ten known honey bee species collected within their natural range distribution. DNA sequences were analyzed using parsimony, distance and maximum likelihood methods to investigate phylogenetic relationships within Apis. The phylogenetic analyses strongly supported the basic topology recoverable from morphometric analysis, grouping the honey bees into three major clusters: giant bees (A. dorsata, A. binghami, and A. laboriosa), dwarf bees (A. andreniformis and A. florea), and cavity-nesting bees (A. mellifera, A. cerana, A. koschevnikovi, A. nuluensis, and A. nigrocincta). However, the clade of Asian cavity-nesting bees included paraphyletic taxa. Exemplars of Apis cerana collected from divergent portions of its range were less related to each other than were sympatric A. cerana, A. nuluensis, and A. nigrocincta taxa. Nucleotide sequence divergence between allopatrically distributed western (A. mellifera) and eastern (A. cerana, A. koschevnikovi, A. nigrocincta, and A. nuluensis) cavity-nesting species, around 18% for the mitochondrial gene and 10-15% for the nuclear intron, suggested an earlier divergence for these groups than previously estimated from morphometric and behavioral studies. This latter finding neccessitates reevaluation of the hypothesized origin of extant European, African, and west Asian Apis mellifera. Sequence divergence between A. laboriosa and A. dorsata was consistent with behavioral data and supports the species status of A. laboriosa.

Amino Acid Sequence↗

PubMed2002

Detection of Y-specific sequences in 122 patients with Turner syndrome: nested PCR is not a reliable method.

The incidence of Y chromosome sequences in patients with Turner syndrome has been evaluated in several studies, and its frequency varied from 0% to 61%, depending on the molecular methodology used. The aim of our study was to screen for Y chromosome sequences in 122 patients with Turner syndrome without cytogenetic evidence of this chromosome. DNA of 100 normal women was also screened and it was used as a negative control. To identify cryptic Y mosaicism, eight regions of Y chromosome were amplified by PCR. In order to increase the sensitivity of Y sequence detection, a nested PCR of the SRY and TSPY genes was also performed. All patients had several stigmata of Turner syndrome and none of them presented with signs of virilization. The most frequent karyotype was 45,X (54.1%), followed by mosaicism involving structural aberration of the X chromosome. There were 12 patients who carried a marker or ring chromosome. First-round PCR identified Y chromosome sequences in only four patients (3%), and all of them had a chromosome mosaicism with at least one cell lineage with a marker chromosome. After nested PCR, 25% of the patients and 14% of the normal women were positive for the presence of Y sequences. Contamination with extraneous genomic DNA was ruled out by microsatellite studies, but we cannot eliminate the possibility of contamination with PCR products, despite careful handling. We conclude that nested PCR overestimated the frequency of Y sequences in patients with Turner syndrome and should be avoided to prevent false positive results, which lead to unnecessary surgical treatment of these patients.

Chromosome Banding↗

PubMed2005

The expression of MAGE and GAGE genes in uterine cervical carcinoma of Korea by RT-PCR with common primers.

BACKGROUND: Melanoma antigen genes (MAGE) and GAGE genes are encoded by genes that are silent in virtually all normal adult tissues but are expressed in tumors from various tissues. These gene products are targets for specific immunotherapy as they are presented by HLA I molecules and recognized by autologous cytotoxic T-lymphocytes. However, the characteristics of these genes, especially in uterine cervical cancer are relatively unknown. PURPOSE: This study evaluated the prevalence of MAGE and GAGE by reverse transcription-polymerase chain reaction (RT-PCR) with common primers and discusses clinical implications in cervical carcinoma. MATERIALS AND METHODS: Fresh tissue from 37 cases of primary squamous cell carcinoma and normal cervical mucosa were evaluated for clinicopathologic parameters including Human Papilloma Virus (HPV)-16,18 infection by PCR, tumor stage by FIGO classification and lymph node involvement. RT-nested PCR for the MAGE and GAGE genes was performed with common primers and DNA sequencing after subcloning was used for identification of PCR products of MAGE. Formalin-fixed paraffin embedded material from the same specimen was analyzed by in situ RT-PCR for MAGE. RESULTS: Expression of MAGE and GAGE was not observed in normal tissues. Eleven out of 37 cases expressed MAGE mRNA (29.7%): analysis of subtypes identified one case of MAGE-1, two cases of MAGE-4b, six cases of MAGE-3, and two unknown subtypes. Thirteen out of 37 cases (35.1%) expressed GAGE mRNA. No significant relationships between expression of these genes and FIGO staging, lymph node metastasis or HPV infection were found. CONCLUSION: Expression of MAGE and GAGE may be involved in the development of uterine cervical carcinoma from intraepithelial neoplasia, although without distinct prognostic significance. MAGE and GAGE genes have the potential to be used as targets for the treatment of uterine cervical carcinoma.

Antigens, Neoplasm↗

PubMed2000

Detection and genotyping of Borrelia burgdorferi sensu lato by polymerase chain reaction.

AIM: To isolate and genotype Borrelia burgdorferi genospecies in serum samples of Croatian patients with erythema migrans. METHODS: DNA isolates from sera of patients with erythema migrans were analyzed by nested polymerase chain reaction (PCR), amplifying a segment of flagellin gene with primers encompassing the conserved region of the gene. To screen PCR products for heterogeneity, we performed single-stranded conformation polymorphism (SSCP) analysis. The samples showing differences in SSCP patterns were sequenced, and the sequence compared in the GeneBank for sequence homology with known Borrelia burgdorferi genospecies. We also constructed phylogenetic tree of all known borrelial sequences. RESULTS: The nested PCR method using specially designed flagellin gene primers, achieved the sensitivity of 10 genome copies (0.01 pg of purified Borrelia burgdorferi DNA from culture) by dilution analysis. The assay specificity was confirmed by amplification of a part of the flagellin gene from different bacterial species. The primer pairs successfully amplified only Borrelia burgdorferi flagellin gene. The genome of Borrelia burgdorferi sensu lato was detected in the sera of all 10 tested patients with erythema migrans. Sequence data and phylogenetic analysis confirmed that all amplified samples belonged to Borrelia afzelii genospecies. CONCLUSION: Phylogenetic tree analysis placed the borrelial isolates together with Borrelia afzelii sequences into a single group. This finding was additionally supported by sequence homology analysis, which produced a homology score of 99%. In patients with erythema migrans who come from the northwest Croatia, an endemic area for Lyme borreliosis, Borrelia afzelii was the cause of skin manifestations of Lyme borreliosis.

Borrelia burgdorferi Group↗

PubMed2005

Development of a highly sensitive method for detection of clarithromycin-resistant Helicobacter pylori from human feces.

Gastric infection of clarithromycin (CAM)-resistant Helicobacter pylori is one of the major causes of failure to eradicate this organism. A noninvasive and useful method for the detection of CAM-resistant H. pylori from human feces by restriction fragment length polymorphism (RFLP)-nested polymerase chain reaction (PCR) targeting the mutation of the 23S rRNA gene that confers CAM-resistance in H. pylori was developed in this study. Our nested PCR method detected DNA of H. pylori in feces with high sensitivity and specificity compared with both an enzyme-linked immunoadsorbent assay (ELISA) of H. pylori in feces and the isolation of H. pylori from gastric biopsy. Furthermore, the results of mutation analysis of the H. pylori 23S rRNA gene amplified from feces completely correlated with both that of the H. pylori 23S rRNA gene amplified from the isolates of gastric biopsy and the susceptibility of H. pylori isolates to CAM. Therefore, our results show that this RFLP/nested PCR method is useful for the accurate diagnosis of CAM-resistant H. pylori infection from feces.

Clarithromycin↗

PubMed2002

Detection of Cryptococcus neoformans DNA in tissue samples by nested and real-time PCR assays.

Two PCR protocols targeting the 18S rRNA gene of Cryptococcus neoformans were established, compared, and evaluated in murine cryptococcal meningitis. One protocol was designed as a nested PCR to be performed in conventional block thermal cyclers. The other protocol was designed as a quantitative single-round PCR adapted to LightCycler technology. One hundred brain homogenates and dilutions originating from 20 ICR mice treated with different azoles were examined. A fungal burden of 3 x 10(1) to 2.9 x 10(4) CFU per mg of brain tissue was determined by quantitative culture. Specific PCR products were amplified by the conventional and the LightCycler methods in 86 and 87 samples, respectively, with products identified by DNA sequencing and real-time fluorescence detection. An analytical sensitivity of 1 CFU of C. neoformans per mg of brain tissue and less than 10 CFU per volume used for extraction was observed for both PCR protocols, while homogenates of 70 organs from mice infected with other fungi were PCR negative. Specificity testing was performed with genomic DNA from 31 hymenomycetous fungal species and from the ustilaginomycetous yeast Malassezia furfur, which are phylogenetically related to C. neoformans. Twenty-four strains, including species of human skin flora like M. furfur and Trichosporon spp., were PCR negative. Amplification was observed with Cryptococcus amylolentus, Filobasidiella depauperata, Cryptococcus laurentii, and five species unrelated to clinical specimens. LightCycler PCR products from F. depauperata and Trichosporon faecale could be clearly discriminated by melting curve analysis. The sensitive and specific nested PCR assay as well as the rapid and quantitative LightCycler PCR assay might be useful for the diagnosis and monitoring of human cryptococcal infections.

Animals↗

PubMedDate not supplied

Comparative effects of DHEA and DHT on gene expression in human LNCaP prostate cancer cells.

BACKGROUND: DHEA is widely used as a dietary supplement in older men. Because DHEA can be converted to androgens or estrogens, such use may promote prostate cancer. In this study, the effects of DHEA were compared with those of DHT using gene expression array profiles in human LNCaP prostate cancer cells. MATERIALS AND METHODS: LNCaP cells were exposed to DHEA (300 nM), DHT (300 nM), or vehicle for 48 h, and mRNA expression was measured using Affymetrix HU-95 gene chips. Gene expression values were sorted in ascending order on the p-values corresponding to the extent of differential RNA expression between control and either hormone treatment. RESULTS: S100 calcium binding protein, neurotensin, 24-dehydrocholesterol reductase, and anterior-gradient 2 homologue were the four most differentially expressed genes (p-values all < 3 x 10(-5)). Nested tests of differential expression revealed lesser effects of DHEA versus DHT treatment (p < 0.01) for the S100 calcium binding protein and neurotensin genes. Microarray findings were confirmed by QRT-PCR. The top 83 genes exhibiting differential expression after DHEA or DHT were used for pathway analysis. DHT decreased expression of more genes involved in intercellular communication, signal transduction, nucleic acid binding and transport, and in structural components, such as myosin and golgin, than DHEA. CONCLUSION: These data revealed consistent, measurable changes in gene expression patterns following treatment of LNCaP prostate cancer cells with DHEA and DHT. Understanding the mechanisms of DHEA versus DHT actions in the prostate may help clarify the separate and interactive effects of androgenic and estrogenic actions in prostate cancer progression.

Adjuvants, Immunologic↗

PubMed1991

Three in-frame N-terminally different proteins are produced from the repressor locus of the Streptomyces bacteriophage phi C31.

The sequence of the repressor locus, c, of the Streptomyces temperate phage, phi C31, was shown previously to contain an open reading frame encoding a 74 kDa protein. Further analysis of the transcriptional and translational products of the c gene shows a more complex pattern of expression. A nest of three in-frame N-terminally different, C-terminally identical proteins of 74, 54 and 42 kDa were found to be expressed from a corresponding nest of transcripts. The repressor proteins were produced in Escherichia coli and the 42 kDa protein was purified, verified by N-terminal sequencing, and used to raise antibody. The antibody cross-reacted in Western blots with the 74, 54 and 42 kDa proteins expressed in E. coli and Streptomyces lividans and from Streptomyces coelicolor phi C31 lysogens. Analysis of transcription of the c gene by S1 mapping and primer extension showed that the nest of transcripts encoding the repressor protein were induced after heat treatment of the cts locus (Sinclair and Bibb, 1989; this paper). Correspondingly, all three of the repressor proteins were induced. In addition to a promoter, cp1, which lies upstream of the 74 kDa open reading frame, the c locus contained at least one internal promoter, cp2, which transcribes DNA encoding the 54 and 42 kDa proteins. Transcripts initiating from cp3 were observed in RNA preparations from S. lividans containing the c gene deleted for cp1 and cp2, but gene fusions using DNA which should contain any putative promoting activity from this region transcriptionally fused to the xylE gene showed very low levels of expression of catechol 2,3 dioxygenase in S. lividans. The 74 kDa protein was not necessary for super-infection immunity. Data described here and current knowledge of the nature of other 'dual start' genes suggest a model for the regulation of lysis versus lysogeny in phi C31.

Bacteriophages↗

PubMed2001

E-cadherin gene mutations in human intrahepatic cholangiocarcinoma.

Deletions or mutations of the E-cadherin gene may result in reduced cell adhesiveness. In particular, conservative point mutations within the N-terminal calcium-binding pocket (including exons 7, 8, and 9) are frequently detected in several cancers and are enough to abolish cell-cell adhesion. There have been no studies on E-cadherin gene mutations in human intrahepatic cholangiocarcinoma (ICC). Human ICCs were therefore investigated for E-cadherin gene mutations within exons 7, 8, and 9. In addition, the relationships were analysed between their mutations and the immunohistochemical expression of E-cadherin, histological grade, and clinicopathological parameters. The E-cadherin gene was analysed in 34 tumours by nested polymerase chain reaction/single-strand conformation polymorphism (PCR/SSCP) followed by DNA sequencing. In four of the 34 cases (11.8%), tumour-restricted mobility shifts were observed; two cases harboured a single shift, one case presented two different mobility shifts, and one case presented three different mobility shifts within exons 7 and 8, encoding extracellular domains of E-cadherin. Polymorphism as previously reported was not identified and all seven new DNA alterations were not present in genomic DNA of non-tumour origin. The E-cadherin gene mutations correlated significantly with down-regulated E-cadherin protein expression and high ICC histological grade. These data suggest that E-cadherin gene mutations in ICC are associated with reduced cell adhesiveness and high histological grade.

Adult↗

PubMed2004

Cloning and molecular characterization of the beta toxin (phospholipase C) gene of Clostridium haemolyticum.

The phospholipase C (PLPC) gene from Clostridium haemolyticum was amplified using the polymerase chain reaction. Primers were selected from a consensus sequence of closely related clostridial PLPC genes and used to amplify an 871-base pair internal segment of the gene. The internal sequence was used to design nested primers that, together with adapter-specific primers, were used to amplify upstream and downstream sequences. The sequences of upstream and downstream segments were aligned with the internal segment to obtain the entire gene sequence. Primers were selected from the aligned sequence, and the entire gene was amplified, and the PCR product was inserted by ligatation into the pCR 2.1 plasmid. An open reading frame that encodes a 399-amino acid protein, containing a 27-amino acid signal sequence, was identified (GenBank Accession Number AF525415). The molecular weight of the active protein was 42869 Da. A 16-amino acid N-terminal sequence, determined by Edman degradation, exactly matched the putative amino acid sequence of the gene product. Together, N-terminal peptide sequencing and tryptic digestion followed by MALDI-ToF mass spectroscopy verified 48% of the amino acid sequences of the active beta toxin. Comparison of the nucleotide and amino acid sequences with Gene-bank databases demonstrated that the beta toxin of C. haemolyticum exhibits high homology with other bacterial PLPCs. The N-terminal portion of the beta toxin contains zinc-binding residues common to clostridial and other bacterial PLPCs, and it shows 34% homology to the N-terminal domain of bovine arachidonate 5-lipoxygenase. The C-terminal domain of the beta toxin protein shows considerable homology with the C-terminal domains of C. novyi type A PLPC, C. perfringens alpha toxin, C. bifermentens PLPC, although the percent identity between the N-terminal regions is much higher overall than that in the C-terminal domain.

Journal Article↗

PubMed2002

Evaluation of the Amazon River delta as a barrier to gene flow for the regional malaria vector, Anopheles aquasalis (Diptera: Culicidae) in northeastern Brazil.

The Neotropical malaria vector Anopheles aquasalis Curry is distributed predominantly along the Atlantic and Pacific Coasts because of its tolerance for breeding in salt water. We tested the hypothesis that the freshwater Amazon River acts as a barrier to gene flow in northeastern Brazil, by examining variation at a 588-nucleotide fragment of the mitochondrial cytochrome oxidase Igene from five populations. We identified 15 haplotypes of which 5 were shared both (1) between sample localities and (2) across the Amazon River Delta. Sequence divergence ranged from 0.0017-0.0272 (average = 0.0137). Estimates of genetic subdivision based on the presence of the Amazon Riverwere greatest within localities (phi = 0.029) and among regions (phi = 0.018), followed by among localities (phi = 0.011), but none were significant. Parsimony, neighbor-joining, and Nested Clade Analyses were used to estimate relationships among populations and infer evolutionary processes. Two phylogenetically distinct clusters of populations were moderately supported by parsimony. Neighbor-joining trees were poorly resolved, thus providing no geographical resolution and no support for the Amazon River as a barrier to migration. Phylogeographic structure as detected by the Nested Clade Analysis was consistent with restricted gene flow coupled with isolation by distance. Taken together, these analyses suggest that the localities within this region of northeastern Brazil constitute a single large population of An. aquasalis that spans the Amazon Delta.

Animals↗

PubMed2004

Direct quantification of Campylobacter jejuni and Campylobacter lanienae in feces of cattle by real-time quantitative PCR.

Campylobacter species are fastidious to culture, and the ability to directly quantify biomass in microbiologically complex substrates using real-time quantitative (RTQ) PCR may enhance our understanding of their biology and facilitate the development of efficacious mitigation strategies. This study reports the use of nested RTQ-PCR to directly quantify Campylobacter jejuni and Campylobacter lanienae in cattle feces. For C. jejuni, the single-copy mapA gene was selected. For C. lanienae, the three-copy 16S rRNA gene was targeted. RTQ-PCR primers were tested alone or they were nested with species-specific primers, and amplification products were detected using the intercalating dye SYBR Green. Nesting did not increase the specificity or sensitivity of C. jejuni quantification, and the limit of quantification was 19 to 25 genome copies ( approximately 3 x 10(3) CFU/g of feces). In contrast, nested RTQ-PCR was necessary to confer specificity on C. lanienae by targeting the 16S rRNA gene. The limit of quantification was 1.8 genome copies ( approximately 250 CFU/g of feces), and there was no discernible difference between the two C. lanienae secondary primer sets evaluated. Detection and quantification of C. jejuni in naturally infested cattle feces by RTQ-PCR were comparable to the results of culture-based methods. In contrast, culturing did not detect C. lanienae in 6 of 10 fecal samples positive for the bacterium and substantially underestimated cell densities relative to nested RTQ-PCR. The results of this study illustrate that RTQ-PCR can be used to directly quantify campylobacters, including very fastidious species, in a microbiologically and chemically complex substrate. Furthermore, targeting of a multicopy universal gene provided highly sensitive quantification of C. lanienae, but nested RTQ-PCR was necessary to confer specificity. This method will facilitate subsequent studies to elucidate the impact of this group of bacteria within the gastrointestinal tracts of livestock and studies of the factors that influence colonization success and shedding.

Animals↗

PubMed2002

Whole CagA gene amplification of Helicobacter pylori and its fingerprinting by restriction fragment length polymorphism.

To set up a method of amplification for the whole CagA gene of Helicobacter pylori and its fingerprinting by restriction fragment length polymorphism (RFLP), nested PCR was employed in combination with TD-PCR to amplify the gene and EcoRI and Hind III were used to generate the RFLP fingerprinting. Target DNA fragments from 13 of 20 samples were successfully amplified and the relevant RFLP fingerprintings were obtained. It is concluded that the method can be used to amplify the whole CagA gene and CagA gene has apparent diversity of RFLP profile.

Antigens, Bacterial↗

PubMed1999

Gene by environment interaction and asthma.

The term interaction has two specific meanings in epidemiology. The first definition is purely a statistical one. Interaction is defined as the coefficient of the product term of two or more risk factors. An alternative definition is a more biologically oriented one in which interaction between two risk factors is defined as their coparticipation in the same causal mechanism of disease development. There are at least five different types of biological gene by environment interaction. It is important to recognize that clinical or epidemiological studies alone are unable to distinguish between these five different types of biological interaction. Asthma is a prototypical complex trait in which gene by environment interaction is important in disease causation. Environmental factors important in asthma include cigarette smoking (active/passive), allergen exposure, viral and bacterial respiratory illness, occupation, and possibly diet. There are two primary study designs that have been utilized for the assessment of gene by environment interaction: a traditional case control study or a nested case control study or a case control study nested within the larger longitudinal cohort. There are three important methodological issues in the assessment of gene by environment interaction. The first is the issue of misspecification of either genotype or environmental exposure or, for that matter, disease status. The second is possible confounding, and the third is the confounding of interaction with dose response. The four most critical factors for determining sample size for gene by environment interaction studies are the odds ratio for the interaction, the prevalence for exposure in the population, the prevalence of the genotype in the population, and the case to control ratio. In general, sample sizes have been too small to detect meaningful gene by environment interactions.

Animals↗

PubMed2000

[Sequence analyzing and genotyping of the gene encoding glutamate rich protein of geographically different Plasmodium falciparum isolates obtained from different malaria endemic areas].

OBJECTIVE: To sequence a gene encoding GLURP and identify the genotypes of geographically different Plasmodium falciparum isolates from Yunnan and Hainan Provinces, China. METHODS: The gene of R2 repeat region of GLURP was amplified by the nested polymerase chain reaction and cloned into T-vector. The nucleotide sequence of the GLURP gene was determined using automatic sequencer (dideoxy chain termination method), and analyzed by DNA Star software. RESULTS: At least 7 different GLURP genotypes ranging from 600 bp to 1,500 bp were found in different P. falciparum isolates from Yunnan and Hainan Provinces. R2 region of GLURP gene consisted of several repeat units, each was composed of 19-20 residues which were shown to be highly conserved. The GLURP gene was also size polymorphic due to differences in the number of repeat units, whereas the repeat sequence was conserved. Sequence analysis showed that DNA sequences and deduced amino acid sequences were highly homologous among the geographically dispersed isolates or various isolates from the same geographical region. No obvious differences were found in the GLURP gene sequences among geographically different isolates. CONCLUSION: The GLURP gene of geographically different P. falciparum isolates is highly conserved and size polymorphic, being useful in searching for malaria vaccine candidate antigen and developing a genotyping method for malaria research.

Amino Acid Sequence↗

PubMed2001

[Gene point mutation in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum].

OBJECTIVE: To investigate the gene point mutation in the dihydrofolate reductase-thymidylate synthase(dhfr) gene of Plasmodium falciparum isolate from Yunnan Province strongly associated with pyrimethamine and cycloguanil resistance. METHODS: Nested PCR and restriction endonuclease digestion were applied to detect the gene mutation using dried blood filter paper collected from the fields in Yunnan Province. RESULTS: Different mutations were found in 4 amino acids at positions 16, 51, 108 and 164 of dhfr gene, particularly, Asn-108 and Ile-51, the mutation frequency being 94.1% and 90.1%, respectively. The frequency of the wild-type genotype (3D7-type) Ser-108 appeared lower (9.1%), while the frequency of the Ala-16 was high(61.8%); the mutation-type was very high, the ratio of HB3-type, 7G8-type/FCR3-type and Cambodian-type was 1:21:7.5. CONCLUSION: The investigation first demonstrated that Plasmodium falciparum Yunnan isolate dihydrofolate reductase-thymidylate synthase gene (dhfr) at positions 16, 51, 108 and 164 exhibited different degrees of point mutation. The frequency of mutation of the 7D8-type involved in pyrimethamine resistance was higher, while that of the FCR3-type involved in cycloquanil resistance was lower.

Animals↗

PubMed2002

Scanning mutagenesis using T4 DNA ligase and short degenerate DNA oligonucleotides containing tri-nucleotide mismatches.

Scanning mutagenesis is an attractive tool for protein structure-function correlation analysis. With one round of this method it is possible to obtain a library containing all possible single-residue mutants of the protein of interest. The practical application of this approach is currently limited by the large number and cost of the required 30-35mer oligonucleotides. As an alternative, we studied the ligation of shorter DNA oligonucleotides (6-11mer) containing a degenerate binding site and a desired mutation mismatch to a nested set of megaprimers annealed to the gene of interest. T4 DNA ligase was able to perform this task, and the obtained ligation products were elongated by DNA polymerase. The effectiveness of ligation depends on the length of the random binding site of the mutagenic oligonucleotide, on its molar excess over the template-primer complex and on the position of the mismatching tri-nucleotide insert with respect to the joining site. The secondary structure of the DNA template close to the joining site also influences the ligation yield. Mismatching oligonucleotides, protected by a 3'-phosphate group, were joined to a nested set of megaprimers, the latter being obtained by a novel procedure called reversible chain termination, i.e., termination of the dsDNA synthesis with ddNTP followed by the subsequent removal of the incorporated ddNMP with exonuclease III. T7 sequenase 2.0 DNA polymerase elongated the ligation products after the 3'-phosphate protection group was removed with T4 polynucleotide kinase, resulting in the incorporation of a specific tri-nucleotide mismatch into dsDNA. This sequence of reactions serves as the basis for a novel scanning mutagenesis procedure.

Bacteriophage T4↗

PubMed2005

A ribosomal RNA gene intergenic spacer based PCR and DGGE fingerprinting method for the analysis of specific rhizobial communities in soil.

A direct molecular method for assessing the diversity of specific populations of rhizobia in soil, based on nested PCR amplification of 16S-23S ribosomal RNA gene (rDNA) intergenic spacer (IGS) sequences, was developed. Initial generic amplification of bacterial rDNA IGS sequences from soil DNA was followed by specific amplification of (1) sequences affiliated with Rhizobium leguminosarum "sensu lato" and (2) R. tropici. Using analysis of the amplified sequences in clone libraries obtained on the basis of soil DNA, this two-sided method was shown to be very specific for rhizobial subpopulations in soil. It was then further validated as a direct fingerprinting tool of the target rhizobia based on denaturing gradient gel electrophoresis (DGGE). The PCR-DGGE approach was applied to soils from fields in Brazil cultivated with common bean (Phaseolus vulgaris) under conventional or no-tillage practices. The community fingerprints obtained allowed the direct analysis of the respective rhizobial community structures in soil samples from the two contrasting agricultural practices. Data obtained with both primer sets revealed clustering of the community structures of the target rhizobial types along treatment. Moreover, the DGGE profiles obtained with the R. tropici primer set indicated that the abundance and diversity of these organisms were favoured under NT practices. These results suggest that the R. leguminosarum-as well as R. tropici-targeted IGS-based nested PCR and DGGE are useful tools for monitoring the effect of agricultural practices on these and related rhizobial subpopulations in soils.

Agriculture↗