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PubMed2003

Bacterial community dynamics during production of registered designation of origin Salers cheese as evaluated by 16S rRNA gene single-strand conformation polymorphism analysis.

Microbial dynamics during processing and ripening of traditional cheeses such as registered designation of origin Salers cheese, an artisanal cheese produced in France, play an important role in the elaboration of sensory qualities. The aim of the present study was to obtain a picture of the dynamics of the microbial ecosystem of RDO Salers cheese by using culture-independent methods. This included DNA extraction, PCR, and single-strand conformation polymorphism (SSCP) analysis. Bacterial and high-GC% gram-positive bacterial primers were used to amplify V2 or V3 regions of the 16S rRNA gene. SSCP patterns revealed changes during the manufacturing of the cheese. Patterns of the ecosystems of cheeses that were provided by three farmers were also quite different. Cloning and sequencing of the 16S rRNA gene revealed sequences related to lactic acid bacteria (Lactococcus lactis, Streptococcus thermophilus, Enterococcus faecium, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Lactobacillus plantarum, and Lactobacillus pentosus), which were predominant during manufacturing and ripening. Bacteria belonging to the high-GC% gram-positive group (essentially corynebacteria) were found by using specific primers. The present molecular approach can effectively describe the ecosystem of artisanal dairy products.

Cheese↗
PubMed1992

Reduced peptide bond cyclic somatostatin based opioid octapeptides. Synthesis, conformational properties and pharmacological characterization.

The conformational and pharmacological properties that result from peptide bond reduction as well as the use of secondary amino acids in a series of cyclic peptides related to the mu opioid receptor selective antagonist D-Phe1-Cys2-Tyr3-D-Trp4-Orn5-Thr6-Pen7+ ++-Thr8-NH2 (IV), have been investigated. Peptide analogues that contain [CH2NH] and [CH2N] pseudo-peptide bonds (in primary and secondary amino acids, respectively) were synthesized on a solid support. Substitution of Tyr3 in IV by the cyclic, secondary amino acid 1,2,3,4-tetrahydroisoquinoline carboxylate (Tic) and of D-Trp4 with D-1,2,3,4-tetrahydro-beta-carboline(D-Tca4), gave peptides 4 and 1, respectively. Both analogues displayed reduced affinities for mu opioid receptors. Conformational analysis based on extensive NMR investigations demonstrated that the backbone conformations of 1 and 4 are similar to those of the potent and selective analogue D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (I), while the conformational properties of the side chains of Tic3 (4) and D-Tca4 (1) resulted in topographical properties that were not well recognized by the mu opioid receptor. Peptide bond modifications were made including (Tyr3-psi[CH2NH]-D-Trp4), 3; (Tyr3-psi[CH2N]-D-Tca4), 2; and (Cys2-psi[CH2N]-Tic3), 6. These analogues showed decreases in their mu opioid receptor affinities relative to the parent compounds IV, 1, and 4, respectively. 1H NMR based conformational analysis in conjunction with receptor binding data led to the conclusion that the reduced peptide bonds in 2, 3, 5, and 6 do not contribute to the process of discrimination between mu and delta opioid receptors, and in spite of their different dynamic behaviors (relative to 1 and 4), they are still capable of attaining similar receptor bound conformations, possibly due to their increased flexibility.

Amino Acid Sequence↗
PubMed2005

[Polymerase chain reaction-single strand conformation polymorphism analysis combined with nucleic acid hybridization to detect ofloxacin-resistant Mycobacterium tuberculosis mutation selected in vitro].

OBJECTIVE: To explore the feasibility of utilizing polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis combined with Southern blot to detect ofloxacin (OFLX)-resistant Mycobacterium tuberculosis, and figure out the boundary between the OFLX-susceptible and resistant Mycobacterium tuberculosis. METHODS: OFLX-resistant Mycobacterium tuberculosis bacilli were induced in vitro and their minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) were determined. The 320 bp segments of gyrA gene were amplified by PCR and their polymorphism was tested by SSCP from H(37)Rv, H(37)Ra and those induced OFLX-resistant strains. To prove the repeatability of PCR-SSCP combined with Southern blot, 36 clinical isolated OFLX-resistant strains were tested. RESULTS: All of the 85 OFLX-resistant mutations involved in this study were identified to have gyrA mutations. The results showed that there was an overlap if OFLX 10 microg/ml was used as a criteria for discriminating susceptible and resistant strains. Compared with the H(37)Ra, the similar single-stranded shift caused by the single-stranded conformation change was viewed in the 36 clinical isolated OFLX-resistant strains by PCR-SSCP combined with Southern hybridization. CONCLUSIONS: PCR-SSCP analysis combined with Southern blot can clearly distinguish OFLX-susceptible from OFLX-resistant strains. The results suggest that as a cut-off point between OFLX-susceptible and low-level OFLX-resistant to Mycobacterium tuberculosis, OFLX concentration of 10 microg/ml is considered to be a better one.

Antitubercular Agents↗
PubMed2005

Ampelomyces mycoparasites from apple powdery mildew identified as a distinct group based on single-stranded conformation polymorphism analysis of the rDNA ITS region.

Pycnidial fungi belonging to the genus Ampelomyces are the most common natural antagonists of powdery mildews worldwide. During a study of the interactions between apple powdery mildew (Podosphaera leucotricha) and Ampelomyces mycoparasites, 52 new Ampelomyces isolates were obtained from P. leucotricha and, in addition, 13 new isolates from other species of the Erysiphaceae in four European countries. Their genetic diversity was screened using single-stranded conformation polymorphism (SSCP) analysis of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA). For comparison, 24 isolates obtained from genetic resource collections or other sources were included in this study. Based on the ITS-SSCP patterns, the isolates were placed in eight groups. The isolates belonged to two types based on their growth in culture. The faster-growing and the slower-growing isolates were included in different SSCP groups. A phylogenetic analysis of the ITS sequences of representatives of these groups confirmed the results obtained with the SSCP method, and showed that the faster-growing isolates do not belong to Ampelomyces as suggested by earlier studies. All the isolates from P. leucotricha fell into a distinct SSCP group of genetically homogeneous isolates. This suggests that Ampelomyces mycoparasites which occur in apple powdery mildew are slightly different from the other Ampelomyces groups which contain mycoparasites from various powdery mildew species. This may be because the main growth period of Ampelomyces mycoparasites in apple powdery mildew is isolated in time from that of Ampelomyces isolates that occur in other species of the Erysiphaceae. P. leucotricha starts its life-cycle early in the season, usually in March-April, while most powdery mildews are active in the same environments only late in the year.

Ascomycota↗
PubMed2004

Single-strand conformation polymorphism analysis of the FMR1 gene in autistic and mentally retarded children in Japan.

Fragile X syndrome is one of the most common causes of mental retardation in males, and patients with fragile X syndrome occasionally develop autism. It is usually caused by an expansion of the trinucleotide repeat in the 5'-untranslated region of the FMR1 gene, but in a small number of patients deletions and point mutations have been identified. We screened all 17 exons of the FMR1 gene for mutations in 90 autistic or mentally retarded children using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis. No mutations were found in 76 male patients. However, one female patient was heterozygous for a normal allele and a mutant allele with an A to C substitution at nucleotide 879 in exon 9. This mutation was not found in 50 controls. Reverse transcription-PCR revealed that a large proportion of the mutant transcripts were spliced aberrantly, causing premature termination of the protein synthesis. Although uncommon, point mutations in the FMR1 gene may be a cause of autism and mental retardation in Japanese patients.

Alternative Splicing↗
PubMed2004

Nonisotopic single-strand conformation polymorphism analysis of sequence variability in ribosomal DNA expansion segments within the genus Trichinella (Nematoda: Adenophorea).

A nonisotopic single-strand conformation polymorphism (SSCP) approach was employed to 'fingerprint' sequence variability in the expansion segment 5 (ES5) of domain IV and the D3 domain of nuclear ribosomal DNA within and/or among isolates and individual muscle (first-stage) larvae representing all currently recognized species/genotypes of Trichinella. In addition, phylogenetic analyses of the D3 sequence data set, employing three different tree-building algorithms, examined the relationships among all of them. These analyses showed strong support that the encapsulated species T. spiralis and T. nelsoni formed a group to the exclusion of the other encapsulated species T. britovi and its related genotypes Trichinella T8 and T9 and T. murrelli, and T. nativa and Trichinella T6, and strong support that T. nativa and Trichinella T6 grouped together. Also, these eight encapsulated members grouped to the exclusion of the nonencapsulated species T. papuae and T. zimbabwensis and the three representatives of T. pseudospiralis investigated. The findings showed that nonencapsulated species constitute a complex group which is distinct from the encapsulated species and supported the current hypothesis that encapsulated Trichinella group external to the nonencapsulated forms, in accordance with independent biological and biochemical data sets.

Animals↗
PubMed2002

Characterization of Salmonella serovars by PCR-single-strand conformation polymorphism analysis.

PCR-restriction fragment length polymorphism (PCR-RFLP) and PCR-single-strand conformation polymorphism (PCR-SSCP) analyses were carried out on the 1.6-kb groEL gene from 41 strains of 10 different Salmonella serovars. Three HaeIII RFLP profiles were recognized, but no discrimination between the serovars could be achieved by this technique. However, PCR-SSCP analysis of the groEL genes of various Salmonella serovars produced 14 SSCP profiles, indicating the potential of this technique to differentiate different Salmonella serovars (interserovar differentiation). Moreover, PCR-SSCP could differentiate strains within a subset of serovars (intraserovar discrimination), as three SSCP profiles were produced for the 11 Salmonella enterica serovar Enteritidis strains, and two SSCP profiles were generated for the 7 S. enterica serovar Infantis and five S. enterica serovar Newport strains. PCR-SSCP has the potential to complement classical typing methods such as serotyping and phage typing for the typing of Salmonella serovars due to its rapidity, simplicity, and typeability.

Bacterial Typing Techniques↗
PubMed1992

Infrequent mutation of p53 gene in human renal cell carcinoma detected by polymerase chain reaction single-strand conformation polymorphism analysis.

Mutation of the p53 gene, which plays an important role in the genesis of diverse human cancers, was investigated in 23 surgical specimens of human renal cell carcinoma using the polymerase chain reaction single-strand conformation polymorphism method of analysis. Only one of the 23 tumors (4.3%) carried a mutated p53 gene, which was present in exons 7-8. Direct DNA sequencing confirmed a point mutation at codon 276 (GCC to CCC) resulting in a substitution of alanine for proline. No specific clinicopathological characteristics were observed in the case with the p53 gene mutation in human renal cell carcinoma. These observations suggest that mutation of the p53 gene is rare and thus does not contribute significantly to the genesis of this tumor.

Amino Acid Sequence↗
PubMed2004

Delineation of Clavispora lusitaniae clinical isolates by polymerase chain reaction-single strand conformation polymorphism analysis of the ITSI region: a retrospective study comparing five typing methods.

Strain delineation of the emerging opportunistic pathogen Clavispora lusitaniae was studied using 12 strains, including two strains of known opposite mating type, CBS 6936 (h+) and CBS 5094 (h-), and 10 strains isolated between 1998 and 2001 from immunocompromized patients. This retrospective study assessed the occurrence of C. lusitaniae subtypes within and among hospitals, and in outpatients who were regularly screened for fungal infections in the course of radio-chemotherapy. Strain typing was accomplished for the first time using single strand conformation polymorphism (SSCP) analysis of amplicons of the ribosomal DNA internal transcribed spacer (ITS) 1 and 2 regions. The results were compared with those produced by three pulsed-field gel electrophoresis (PFGE) methods and PCR fingerprinting with the minisatellite-specific primer M13. Karyotyping separated 7-9 chromosomes, not 6-8 as previously reported. Pulsotyping of SfiI and NotI digested chromosomes grouped isolates in five and four distinct clusters, respectively. All methods revealed strain heterogeneity, though not as extensive as previously recorded. SSCP analysis of the ITS1 region generated five subtypes, based on a sequencing-confirmed nucleotide polymorphism. The discriminatory power of this method was high. All strains displayed a homogeneous SSCP pattern for the ITS2 region. ITS1 PCR-SSCP appears to allow rapid and reliable delineation of C. lusitaniae strains. Pending examination of a larger sample size and interlaboratory study, this protocol can be recommended for rapid prospective identification of hospital outbreaks.

Amino Acid Sequence↗
PubMed1997

Detection of rifampin resistance by single-strand conformation polymorphism analysis of cerebrospinal fluid of patients with tuberculosis of the central nervous system.

Mutations in a 69-bp region of the rpoB gene of Mycobacterium tuberculosis are associated with rifampin resistance (Rif[r]). These have been detected with mycobacterial DNA extracted from bacterial suspensions or respiratory specimens that were acid-fast smear positive. We experimented with a strategy for the rapid detection of Rif(r) in cerebrospinal fluid (CSF) samples. The strategy involves the amplification of the 69-bp region of rpoB by means of PCR and the identification of nucleotide mutations by single-strand conformation polymorphism (SSCP) analysis of the amplification products. Sixty-five CSF specimens collected from 29 patients (19 patients were coinfected with human immunodeficiency virus) with culture or autopsy-confirmed (22 patients) or highly probable (7 patients) tuberculosis of the central nervous system (CNS-TB) were processed. Amplified products suitable for evaluation by SSCP analysis were obtained from 37 CSF specimens from 25 subjects (86.2%). PCR-SSCP of CSF correctly identified the rifampin susceptibility phenotype of isolates from all 17 patients for whom the results of susceptibility tests carried out with strains cultured from CSF or respiratory samples were available. Moreover, this assay revealed the rifampin susceptibility genotype of isolates from the eight patients (three patients with culture-confirmed CNS-TB and five patients in whom CNS-TB was highly probable) for whom no susceptibility test results were available; the PCR-SSCP data obtained for these patients were concordant with the outcome after a standard antituberculosis treatment. The evolution of a mutation in the rpoB gene was documented in a patient during the course of treatment. PCR-SSCP analysis of CSF seems to be an efficacious method of predicting Rif(r) and would reduce the time required for susceptibility testing from approximately 4 to 8 weeks to a few days.

AIDS-Related Opportunistic Infections↗
PubMed1998

Genotyping metabolic polymorphisms in a cohort of Caucasians and single strand conformation polymorphism analysis of point mutations in human hprt exons 7 and 8.

In genetic toxicology, the main fields of applications of the polymerase chain reaction (PCR) with subsequent electrophoretic characterization of amplificates include genotyping polymorphisms in the xenobiotic metabolism and mutant analysis. To assess the role of the individual sets of biotransformation enzymes for the internal dose resulting from xenobiotic exposure, we investigated blood samples from 69 healthy donors for the occurrence of known genetic polymorphisms in the xenobiotic metabolizing enzymes N-acetylaminotransfrase II (NAT2), glutathione-S-transferase (GST) mu and theta, and several cytochromes P450 (CYP), namely CYP1A1, CYP2E1 and CYP2A6. Using single strand conformation polymorphism (SSCP) analysis, five known single base substitutions located in the middle portion of 144 bp amplificates comprising exons 7 and 8 of the human hypoxanthine guanine phosphoribosyl transferase (hprt) cDNA, were clearly distinguished from wild type and from each other. Biomagnetic strand separation assigned the slower migrating single strand bands to the biotinylated sense strands.

Aryl Hydrocarbon Hydroxylases↗
PubMed2006

Optimization of capillary array electrophoresis single-strand conformation polymorphism analysis for routine molecular diagnostics.

Mutation screening is widely used for molecular diagnostics of inherited disorders. Furthermore, it is anticipated that the present and future identification of genetic risk factors for complex disorders will increase the need for high-throughput mutation screening technologies. Capillary array electrophoresis (CAE) SSCP analysis is a low-cost, automated method with a high throughput and high reproducibility. Thus, the method fulfills many of the demands to be met for application in routine molecular diagnostics. However, the need for performing the electrophoresis at three temperatures between 18 degrees C and 35 degrees C for achievement of high sensitivity is a disadvantage of the method. Using a panel of 185 mutant samples, we have analyzed the effect of sample purification, sample medium and separation matrix on the sensitivity of CAE-SSCP analysis to optimize the method for molecular diagnostic use. We observed different effects from sample purification and sample medium at different electrophoresis temperatures, probably reflecting the complex interplay between sequence composition, electrophoresis conditions and sensitivity in SSCP analysis. The effect on assay sensitivity from three different polymers was tested using a single electrophoresis temperature of 27 degrees C. The data suggest that a sensitivity of 98-99% can be obtained using a 10% long chain poly-N,N-dimethylacrylamide polymer.

Acrylamides↗
PubMed1992

Sensitive detection of p53 gene mutations in esophageal endoscopic biopsy specimens by cell sorting combined with polymerase chain reaction single-strand conformation polymorphism analysis.

For the rapid and sensitive detection of p53 gene mutations in esophageal endoscopic biopsy specimens, we combined cell sorting with the polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analysis. Mutations in exons 5-8 of the p53 gene were investigated by PCR-SSCP analysis using 10(3) sorted nuclei obtained from each endoscopic biopsy specimen of 16 patients with esophageal cancer. DNAs extracted from their respective surgical specimens were investigated by a conventional method of PCR-SSCP analysis. Mutations in the biopsy specimens were detected in 6 of the 12 aneuploid tumors but in none of the 4 diploid tumors. After tumor cell enrichment by cell sorting, one mutation in exon 8 became apparent, which could not be detected from the surgical specimen by a conventional method of PCR-SSCP analysis. This method should improve the sensitivity of detecting p53 gene mutations, and provides additional information concerning the DNA ploidy pattern in the tumors.

Aged↗
PubMed1993

Detection of clonal immunoglobulin gene rearrangements by polymerase chain reaction amplification and single-strand conformational polymorphism analysis.

Analysis of immunoglobulin gene rearrangements by Southern blotting is a sensitive and specific method for detecting B cell malignancies but requires a relatively large amount of intact DNA. It cannot be utilized in many cases where only a small amount of tissue is available or where the tissue has been fixed. This report demonstrates that polymerase chain reaction (PCR) amplification in conjunction with single-strand conformational polymorphism (SSCP) analysis can be utilized to detect clonal immunoglobulin heavy chain (IgH) gene rearrangements. IgH gene rearrangements from a series of frozen or formalin-fixed B cell malignancies were PCR-amplified using oligonucleotide primers, based upon consensus sequences in the IgH variable and joining regions. Analysis of the single-stranded PCR products on nondenaturing polyacrylamide gels revealed discrete SSCPs corresponding to the malignant B cells. These SSCPs were detectable when the malignant cells represented as few as 0.2% of the total mononuclear cells in peripheral blood. PCR amplification in conjunction with SSCP analysis thus provides a sensitive and specific method to detect clonal IgH rearrangements from minute amounts of fresh, frozen, or fixed tissue.

Base Sequence↗
PubMed2001

Separation of DNA fragments and single strand conformation polymorphism analysis in bare capillaries using poly(acrylamide-dimethylacrylamide) as a separation medium.

A short chain poly(acrylamide-dimethylacrylamide) (PADMA) was synthesized in aqueous phase using isopropanol as a chain transfer agent, and was characterized according to the chemical composition and molecular mass. This polymer can form a stable dynamic coating on the inner surface of the capillary, thereby suppressing the electroosmotic flow and DNA-capillary wall interaction. The sieving medium has low viscosity and capillary filling with this medium and medium replacement were conveniently carried out by commercial capillary electrophoresis instruments. The effects of components and concentration of copolymers on the separation of DNA fragments were investigated. Highly efficient separation of DNA fragments, successful single strand conformation polymorphism (SSCP) analysis and good reproducibility of the migration time were obtained in bare capillaries using these copolymers as sieving media. Our preliminary results demonstrate that PADMA will become an alternative matrix for DNA separation by capillary electrophoresis.

Acrylamides↗
PubMed2001

Identification of Mycobacterium species by multiple-fluorescence PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene.

Identification of mycobacteria to the species level by growth-based methodologies is a process that has been fraught with difficulties due to the long generation times of mycobacteria. There is an increasing incidence of unusual nontuberculous mycobacterial infections, especially in patients with concomitant immunocompromised states, which has led to the discovery of new mycobacterial species and the recognition of the pathogenicity of organisms that were once considered nonpathogens. Therefore, there is a need for rapid and sensitive techniques that can accurately identify all mycobacterial species. Multiple-fluorescence-based PCR and subsequent single-strand conformation polymorphism (SSCP) analysis (MF-PCR-SSCP) of four variable regions of the 16S rRNA gene were used to identify species-specific patterns for 30 of the most common mycobacterial human pathogens and environmental isolates. The species-specific SSCP patterns generated were then entered into a database by using BioNumerics, version 1.5, software with a pattern-recognition capability, among its multiple uses. Patient specimens previously identified by 16S rRNA gene sequencing were subsequently tested by this method and were identified by comparing their patterns with those in the reference database. Fourteen species whose SSCP patterns were included in the database were correctly identified. Five other test organisms were correctly identified as unique species or were identified by their closest relative, as they were not in the database. We propose that MF-PCR-SSCP offers a rapid, specific, and relatively inexpensive identification tool for the differentiation of mycobacterial species.

DNA Primers↗
PubMed2003

Genetic diversity among Borrelia strains determined by single-strand conformation polymorphism analysis of the ospC gene and its association with invasiveness.

Lyme borreliosis (LB) is a tick-borne spirochetal infection caused by three Borrelia species: Borrelia afzelii, B. garinii, and B. burgdorferi sensu stricto. LB evolves in two stages: a skin lesion called erythema migrans and later, different disseminated forms (articular, neurological, cardiac.). Previous research based on analysis of ospC sequences allowed the definition of 58 groups (divergence of <2% within a group and >8% between groups). Only 10 of these groups include all of the strains isolated from disseminated forms that are considered invasive. The aim of this study was to determine whether or not invasive strains belong to restricted ospC groups by testing human clinical strains isolated from disseminated forms. To screen for ospC genetic diversity, we used single-strand conformation polymorphism (SSCP) analysis. Previously known ospC sequences from 44 different strains were first tested, revealing that each ospC group had a characteristic SSCP pattern. Therefore, we studied 80 disseminated-form isolates whose ospC sequences were unknown. Of these, 28 (35%) belonged to previously known invasive groups. Moreover, new invasive groups were identified: six of B. afzelii, seven of B. garinii, and one of B. burgdorferi sensu stricto. This study confirmed that invasive strains are not distributed among all 69 ospC groups but belong to only 24 groups. This suggests that OspC may be involved in the invasiveness of B. burgdorferi.

Antigens, Bacterial↗
PubMed1994

Double- and single-strand conformation polymorphism analysis of point mutations and short tandem repeats.

Single point mutations in small DNA fragments and single unit differences in simple repetitive DNA can be detected as double-strand conformation polymorphisms using polyacrylamide gel electrophoresis, with and without sodium dodecyl sulfate, even at temperatures as low as 3 degrees C. Changes in a single base are distinguished by means of the analysis of the heteroduplexes, and changes in more than two bases can be distinguished in both homoduplexes and heteroduplexes. Polymerase chain reaction (PCR) conditions can be designed not only to amplify homoduplexes, heteroduplexes and single-strand DNA at the same time, but also to focus the analysis on either single-strand conformation polymorphism (SSCP) or double-strand conformation polymorphism (DSCP). DSCP seems to be advantageous in typing DNA polymorphisms or mutations in loci with few variants, but, because it is necessary to have a simple pattern of all possible combinations of the alleles, it is not as advantageous in typing systems with many variants.

Base Sequence↗