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At least 1,387 records · Page 77Linked to original sources
PubMed2005

Rapid investigation of hepatitis A virus outbreak by single strand conformation polymorphism analysis.

Investigation of hepatitis A virus (HAV) outbreaks often implies nucleotide sequence analysis. As an alternative method for the identification of related strains, single strand conformation polymorphism method (SSCP) was compared to sequence analysis. Twenty-three strains from sporadic and outbreak cases were studied retrospectively. SSCP, sequence identity and phylogenetic analyses were conducted on a 267 bp fragment of the VP1-2A variable region. The results of SSCP pattern comparison and sequence identity were highly correlated (r = 0.92, P < 0.001). If SSCP showed similar patterns, the VP1-2A fragments had a high and significant probability to have a sequence identity over 99.6%. Results were concordant for outbreak strains. The only discordant result concerned a cluster of three sporadic cases evidenced by phylogenetic analysis while SSCP showed similar patterns for only two of these three cases. A prospective SSCP analysis of a recent HAV outbreak confirmed the reliability of this technique. SSCP may thus provide a rapid and cost-effective tool for preliminary investigation of HAV outbreaks, before undertaking exhaustive nucleotide sequence analysis.

Adolescent↗
PubMed1994

Polymerase chain reaction-single strand conformation polymorphism analysis of the p53 gene in paraffin-embedded surgical material from human renal cell carcinomas.

p53 tumour suppressor gene mutations were studied in 118 renal cell carcinomas using paraffin-embedded surgical material. Optimal results were obtained with analysis of exon lengths between 150 and 200 base pairs for polymerase chain reaction. Single strand conformation polymorphism and sequencing analysis revealed only two point mutations (2/118, 2%): one involving codon 135; TGC-->TTC (cysteine-->phenylalanine) and the other codon 175; CGC-->CAC (arginine-->histidine). Both of these cases were classified as granular cell subtype on microscopic observation. The data suggest that the p53 tumour suppressor gene is not related to tumour initiation, promotion, or progression of renal cell carcinomas. However, there is the possibility that granular cell type carcinomas may have a different genetic background from clear cell type renal neoplasms.

Base Sequence↗
PubMed1993

Direct cycle sequencing of mutated alleles detected by PCR single-strand conformation polymorphism analysis.

A rapid protocol for direct sequencing of a mutated allele, detected by combining polymerase chain reaction with single-strand conformation polymorphism (PCR-SSCP) analysis and cycle sequencing using a thermal cycler, is described. End-labeled radioactive primers were used both for PCR-SSCP analysis for the detection of p53 gene mutation and for cycle sequencing using delta Taq Version 2.0 DNA Polymerase. The point mutations along the various exons of the p53 gene can be rapidly determined by this sequencing method. This protocol requires only a small amount of DNA template (0.1 microgram) and produces sequencing images with low backgrounds and very uniform band intensity. It has also been used successfully in the study of other gene mutations including ras and NF-1 (neurofibromatosis 1) genes.

Alleles↗
PubMed2003

Chloroplast inheritance patterns in Actinidia hybrids determined by single stranded conformation polymorphism analysis.

The inheritance patterns of the chloroplast genomes of the Actinidia hybrids A. eriantha (male parent) x A. chinensis (female parent) and A. chinensis (male parent) x A. melanandra (female parent) were analyzed using single-strand conformation polymorphism (SSCP) analysis of the trnL-trnF and psbA-trnH intergenic spacers. This showed that the artificial hybrids between A. eriantha and A. chinensis all had the haplotype of their male parent. Alignment of the sequences of A. eriantha and A. chinensis revealed four substitutions and one insertion (GATTC) in trnL-trnF and two substitutions in psbA-trnH. In contrast, the haplotypes of the artificial hybrids between A. chinensis and A. melanandra had the same patterns as their female parent. Alignment of the entire region of A. chinensis and A. melanandra revealed 12 substitutions: 1 in trnL-trnF and 11 in psbA-trnH. However, no sequence variation in the trnL-trnF and psbA-trnH intergenic spacers was found. We have developed a simple screening method for detecting the inheritance patterns of Actinidia chloroplast DNA haplotypes using SSCP analysis of the trnL-trnF and psbA-trnH intergenic spacers. Our findings indicate that the inheritance of the chloroplast genome in Actinidia hybrids differs according to the species selected.

Actinidia↗
PubMed1992

Cloning and polymerase chain reaction-single-strand conformation polymorphism analysis of anonymous Alu repeats on chromosome 11.

We have shown that many of the Alu repeats found in the GenBank database are polymorphic and that this polymorphism can be detected by a simple technique, single-strand conformation polymorphism (SSCP) analysis, after polymerase chain reaction (PCR) amplification of each repeat from DNA of individuals. Here, we describe a method for collecting many anonymous Alu repeats and their flanks in a chromosome-specific phage library and cloning them into plasmids. The flanking single-copy sequences of each repeat in the plasmid were then determined, and 20mer to 30mer segments of these sequences were used as primers for the PCR-SSCP analysis. Many new polymorphic DNA markers on chromosome 11 were obtained with this method. These markers can also serve as sequence-tagged sites for physical mapping of the genome.

Bacteriophages↗
PubMed1999

Fluorescence-based single-strand conformation polymorphism analysis of the low density lipoprotein receptor gene by capillary electrophoresis.

We describe here a new method to screen for unknown mutations in the low density lipoprotein (LDL) receptor gene by the use of capillary electrophoresis in single-strand conformation polymorphism (SSCP) analysis. To analyze the promoter and all 18 exons, 20 different amplification reactions were necessary. For each polymerase chain reaction (PCR), the forward and reverse primers were 5' fluorescent-labelled with FAM and HEX, respectively. To test the accuracy of the newly developed method, 61 genetic variants distributed in 16 exons were analyzed. Under identical electrophoresis conditions (13 kV, 30 degrees C, 30 min), 59 mutations were detected by a distinct abnormal SSCP pattern. The two remaining mutations showed only slight abnormalities, which could be amplified by increasing the electrophoresis temperature. The high accuracy, the degree of automation and the speed of analysis make fluorescence-based SSCP analysis with capillary electrophoresis ideal for rapid mutation screening and the technique is well-suited for clinical applications.

Base Sequence↗
PubMed2003

Genetic variability within Borrelia burgdorferi sensu lato genospecies established by PCR-single-strand conformation polymorphism analysis of the rrfA-rrlB intergenic spacer in ixodes ricinus ticks from the Czech Republic.

In Europe the Borrelia burgdorferi sensu lato complex is represented by five distinct genospecies: Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana, and Borrelia lusitaniae. These taxonomic entities are known to differ in their specific associations with vertebrate hosts and to provoke distinct clinical manifestations in human patients. However, exceptions to these rules have often been observed, indicating that strains belonging to a single genospecies may be more heterogeneous than expected. It is, therefore, important to develop alternative identification tools which are able to distinguish Borrelia strains not only at the specific level but also at the intraspecific level. DNA from a sample of 370 Ixodes ricinus ticks collected in the Czech Republic was analyzed by PCR for the presence of a approximately 230-bp fragment of the rrfA-rrlB intergenic spacer of Borrelia spp. A total of 20.5% of the ticks were found to be positive. The infecting genospecies were identified by analyzing the amplified products by the restriction fragment length polymorphism (RFLP) method with restriction enzyme MseI and by single-strand conformation polymorphism (SSCP) analysis. The two methods were compared, and PCR-SSCP analysis appeared to be a valuable tool for rapid identification of spirochetes at the intraspecific level, particularly when large samples are examined. Furthermore, by using PCR-SSCP analysis we identified a previously unknown Borrelia genotype, genotype I-77, which would have gone unnoticed if RFLP analysis alone had been used.

Animals↗
PubMed1992

Quantitative analysis of cyclic beta-turn models.

The beta-turn is a frequently found structural unit in the conformation of globular proteins. Although the circular dichroism (CD) spectra of the alpha-helix and beta-pleated sheet are well defined, there remains some ambiguity concerning the pure component CD spectra of the different types of beta-turns. Recently, it has been reported (Hollósi, M., Kövér, K.E., Holly, S., Radics, L., & Fasman, G.D., 1987, Biopolymers 26, 1527-1572; Perczel, A., Hollósi, M., Foxman, B.M., & Fasman, G.D., 1991a, J. Am. Chem. Soc. 113, 9772-9784) that some pseudohexapeptides (e.g., the cyclo[(delta)Ava-Gly-Pro-Aaa-Gly] where Aaa = Ser, Ser(OtBu), or Gly) in many solvents adopt a conformational mixture of type I and the type II beta-turns, although the X-ray-determined conformation was an ideal type I beta-turn. In addition to these pseudohexapeptides, conformational analysis was also carried out on three pseudotetrapeptides and three pseudooctapeptides. The target of the conformation analysis reported herein was to determine whether the ring stress of the above beta-turn models has an influence on their conformational properties. Quantitative nuclear Overhauser effect (NOE) measurements yielded interproton distances. The conformational average distances so obtained were interpreted utilizing molecular dynamics (MD) simulations to yield the conformational percentages. These conformational ratios were correlated with the conformational weights obtained by quantitative CD analysis of the same compounds. The pure component CD curves of type I and type II beta-turns were also obtained, using a recently developed algorithm (Perczel, A., Tusnády, G., Hollósi, M., & Fasman, G.D., 1991b, Protein Eng. 4(6), 669-679). For the first time the results of a CD deconvolution, based on the CD spectra of 14 beta-turn models, were assigned by quantitative NOE results. The NOE experiments confirmed the ratios of the component curves found for the two major beta-turns by CD analysis. These results can now be used to enhance the conformational determination of globular proteins on the basis of their CD spectra.

Amino Acid Sequence↗
PubMed2002

High-throughput, high-sensitivity genetic mutation detection by tandem single-strand conformation polymorphism/heteroduplex analysis capillary array electrophoresis.

We present the first optimization of linear polyacrylamide (LPA)-based DNA separation matrixes for an automated tandem microchannel single-strand conformation polymorphism (SSCP)/heteroduplex analysis (HA) method, implemented in capillary arrays dynamically coated with poly(N-hydroxyethylacrylamide) (polyDuramide). An optimized protocol for sample preparation allowed both SSCP and HA species to be produced in one step in a single tube and distinguished in a single electrophoretic analysis. A simple, two-color fluorescent sample labeling and detection strategy enabled unambiguous identification of all DNA species in the electropherogram, both single- and double-stranded. Using these protocols and a panel of 11 p53 mutant DNA samples in comparison with wild-type, we employed high-throughput capillary array electrophoresis (CAE) to carry out a systematic and simultaneous optimization of LPA weight-average molar mass (Mw) and concentration for SSCP/HA peak separation. The combination of the optimized LPA matrix (6% LPA, Mw 600 kDa) and a hydrophilic, adsorbed polyDuramide wall coating was found to be essential for resolution of CAE-SSCP/HA peaks and yielded sensitive mutation detection in all 11 p53 samples initially studied. A larger set of 32 mutant DNA specimens was then analyzed using these optimized tandem CAE-SSCP/HA protocols and materials and yielded 100% sensitivity of mutation detection, whereas each individual method yielded lower sensitivity on its own (93% for SSCP and 75% for HA). This simple, highly sensitive tandem SSCP/HA mutation detection method should be easily translatable to electrophoretic analyses on microfluidic devices, due to the ease of the capillary coating protocol and the low viscosity of the matrix.

Electrophoresis, Capillary↗
PubMed2005

Technical note: determination of alleles of the ovine PRNP gene using PCR-single-strand conformational polymorphism analysis.

Susceptibility to scrapie in sheep is linked to variation at codons 136, 154, and 171 in the host prion protein gene (PRNP). A number of techniques are available for detecting these polymorphisms, but none allow for a rapid and accurate determination of genotype. Here we describe PCR coupled with single-strand conformational polymorphism (SSCP) analysis, which allows for the accurate identification of ovine PRNP alleles. A gene region including codons 136 to 171 was amplified by PCR, and the amplimers were then denatured and subjected to electrophoresis in a nondenaturing polyacrylamide gel. Nine unique SSCP patterns, representing nine different alleles of the ovine PRNP gene, could be resolved. A new polymorphism (I/T) at codon 142 also was detected. The profiles produced by SSCP allowed for the accurate differentiation of PRNP alleles and could be employed to genotype PRNP in sheep.

Alleles↗
PubMed1995

Molecular subtyping of human T-cell lymphotropic virus type 2 by single-strand conformation polymorphism analysis. Retrovirus Epidemiology Donor Study Group.

Molecular subtyping of human T-cell lymphotropic virus type 2 (HTLV-2) by the currently used method of restriction fragment length polymorphism analysis may not be sufficiently discriminatory for transmission studies because of the predominance of single restriction types in various HTLV-2-infected populations. The utility of single-strand conformations polymorphism (SSCP) analysis was evaluated as a tool to improve the sensitivity of the subtyping of HTLV-2. The assay was designed to target a highly variable region in the long terminal repeat and was shown to be able to detect single nucleotide changes in cloned HTLV-2 sequences. Analysis of 52 HTLV-2 samples, of which 32 were from 16 sex partner pairs (16 males, 16 females), showed nine different SSCP patterns. Identical SSCP results were obtained for each of the 16 couples, suggesting the presence of similar viral genotypes and, therefore, supporting the likelihood of sexual transmission of HTLV-2 in each of these couples. Furthermore, SSCP analysis of seven HTLV-2 samples of the same restriction type (b5) showed five different SSCP patterns. Nucleotide sequencing of two samples with distinct SSCP patterns confirmed the sequence differences. SSCP provides a facile and discriminatory tool for the differentiation of HTLV-2 strains, including those previously indistinguishable by restriction fragment length polymorphism.

Base Sequence↗
PubMed1994

Effect of non-tumor cell contamination on detection of p53 gene mutations in human gastric cancer cells by polymerase chain reaction single-strand conformation polymorphism analysis.

BACKGROUND: We have previously studied p53 gene mutations in 25 primary gastric cancer tissues by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis for exon 4-8 and immunohistochemical staining with anti-p53 antibody. In four cases, the discrepancy of the results was observed between the two methods. In one case positive by PCR-SSCP but negative by immunohistochemical staining, the mutation was silent. In three cases, the p53 gene mutations were detected only by immunohistochemical staining. This discrepancy may be due to the contamination of the samples by cells without p53 gene mutation, such as non-tumor cells. This study was conducted to investigate the sensitivity of PCR-SSCP analysis to p53 gene mutations when the sample was contaminated with non-tumor cells. METHODS: Genomic DNA was extracted by the digestion with proteinase K and phenol-chloroform-ethanol method from two human gastric adenocarcinoma cell lines, MKN-45 and KATO III. To investigate the sensitivity of PCR-SSCP, DNA extracted from cancer cells was mixed with DNA obtained from normal gastric mucosal cells at various ratios. PCR-SSCP analysis for exon 4-8 of the p53 gene was performed with the mixed DNA samples. RESULTS: In KATO III, no PCR products were generated in exon 4-8 of the p53 gene by PCR, suggesting that both alleles from exon 4-8 of the p53 gene were deleted. In MKN-45, the mobility shift was observed in exon 4. Therefore, the effect of non-tumor cell contamination on the detection of p53 gene mutations was conducted using MKN-45 and normal gastric mucosal cells. In the mixed DNA samples of MKN-45 and normal gastric mucosal cells, an extra band with the migration similar to that of MKN-45 was found in the samples of 1:8 dilution or less, while no extra band was grossly detectable in DNA of normal gastric mucosal cells and in the samples of more than 1:16 dilution. CONCLUSIONS: These results suggest that the detection of p53 mutations by PCR-SSCP analysis may be underestimated in samples contaminated by a large number of non-tumor cells.

Base Sequence↗
PubMed2002

Increasing the sensitivity of single-strand conformation polymorphism analysis of the LDLR gene mutations in brazilian patients with familial hypercholesterolemia.

Mutations in the low-density lipoprotein receptor (LDLR) gene cause familial hypercholesterolemia (FH), one of the most common single gene disorders. It is thought that FH affects approximately 1 of 500 individuals in most populations. Single-strand conformation polymorphism (SSCP) analysis is widely used to detect mutations in the LDLR gene. However, several factors such as temperature, pH, running time, gel composition and size of the DNA fragments can influence its sensitivity. We have optimized the electrophoretic conditions to screen mutations in the promoter region and exons 1-18 of the LDLR gene by varying temperature (5 degrees C, 8 degrees C, 12 degrees C and 15 degrees C), voltage (300 to 600 V), and running time (1 to 4 hours) in the semi-automated GenePhor system (Amersham Biosciences). The efficiency of the method was evaluated by using 30 positive controls (DNA samples with mutations and polymorphisms in the LDLR gene, previously characterized) and DNA samples from 90 Brazilian patients with FH. Our results show that the use of two temperatures (5 degrees C and 15 degrees C) in combination with other optimized conditions resulted in high mutation detection rate (97%), which was considered appropriate for routine screening. Therefore, this strategy could be useful for the diagnosis of genetic disorders, cancer, and for pharmacogenetic studies.

Adolescent↗
PubMed2004

Single-strand conformational polymorphism analysis of the ribosomal internal transcribed spacer 1 for rapid species identification within the genus Pythium.

Single-strand conformational polymorphism (SSCP) of the ribosomal internal transcribed spacer 1 (ITS-1) was characterized for 58 isolates of Pythium, representing 41 species from the five groups of Plaats-Niterink. Thirty-one species each produced a distinct SSCP pattern. Three species produced more than one unique pattern, corresponding to morphological subgrouping. The remaining seven species produced three distinct patterns with two or three morphologically similar species sharing a pattern. A successful blind test with four samples and the identification of eight previously unknown isolates from irrigation water demonstrated the reliability of this technique for species identification. Each SSCP pattern was defined and described by the positions of the top and bottom bands and the number of bands in between, which allows laboratories to use this technique without need to access the type isolates of Pythium species.

DNA, Algal↗
PubMed1992

Detection of p53 gene mutations in human ovarian and endometrial cancers by polymerase chain reaction-single strand conformation polymorphism analysis.

The presence of mutations in the p53 gene was examined in ovarian cancers by a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. The primers were designed to amplify exons 5 through 9 that contain phylogenetically conserved domains of the p53 gene. Mutations were detected in 5 out of 10 cases, one of which contained a deletion in the second allele. A single base substitution was detected in 4 cases at codons 162, 175, 205 and 273 and a single base insertion in one case within codon 315. A high frequency of p53 mutations in ovarian cancers and lack of mutation in 6 benign ovarian tumors and 2 normal ovaries suggested that the mutation of the p53 gene was associated with the genesis and/or progression of ovarian cancer. In 1 of 7 endometrial cancers, two mutations at codons 239 and 254 were detected.

Alleles↗
PubMed2001

Detection of avocado sunblotch viroid variants using fluorescent single-strand conformation polymorphism analysis.

A specific reverse transcription-polymerase chain reaction (RT-PCR) protocol has been developed for routine detection of avocado sunblotch viroid (ASBVd). Modifications in this diagnostic technique were made to enable fluorescent detection and variant identification using automated capillary electrophoresis (CE) and fluorescent single-strand conformation polymorphism (SSCP) analysis. Sixteen sequence variants characterized in a previous study were analyzed using CE-SSCP on two ABI 310 Genetic Analyzers. Significant differences were detected between data obtained from the two ABI 310 Genetic Analyzers indicating that an internal control must be run concurrently with the samples. The 16 variants could be classified into 11 groups based on the SSCP patterns. The statistical analysis of the migration rate data provided support for the visual differences in SSCP patterns. The use of SSCP in the ASBVd assay is easily accomplished and gives an estimate of the number of variants in crude samples extracted from infected avocado plants.

Base Sequence↗
PubMed1996

Genetic characterization of protein C deficiency in Japanese subjects using a rapid and nonradioactive method for single-stand conformational polymorphism analysis and a model building.

We studied the molecular basis of protein C deficiency in 28 Japanese families including 4 asymptomatic families. Two showed a decreased level of function with a normal antigen concentration consistent with type II protein C deficiency and the remaining 26 showed type I deficiency with decreases in both function and antigen level. All the exons and intron/exon junctions of the protein C gene were studied using a strategy combining polymerase chain reaction (PCR) amplification and rapid nonradioactive single-strand conformational polymorphism (SSCP) analysis. The PCR-amplified fragments with aberrant migration on SSCP analysis were sequenced. We identified 11 missense mutations, 1 nonsense mutation, 2 neutral polymorphisms, 1 frameshift deletion, 1 inframe deletion, and 1 splice site mutation. We also identified two different rare mutations in the 5'-untranslated region in the protein C gene that may be responsible for the phenotype. Of these molecular defects, ten were novel. From the results of genetic analysis of 47 Japanese families with protein C deficiency reported in this and previous studies, Phe139Val and Met364Ile substitutions and a G8857 deletion were only found in Japanese subjects and seem to be a founder effect. In contrast, Arg169Trp and Val297Met substitutions, both occurring at CG dinucleotides, were commonly observed in not only Japanese but also Western populations, indicating that these are hot spots for mutation in the protein C gene. These molecular defects were found in 22 families in total, accounting for 47% of Japanese families with protein C deficiency. The structural models of the second EGF and protease domains of activated wild-type and mutant human protein C suggest a possible substrate binding exosite on two loops; one from amino acid position 349 to 357 and the other from position 385 to 388, both of which are close to each other in the three-dimensional model.

Amino Acid Sequence↗
PubMed1992

Use of single strand conformation polymorphism analysis to detect point mutations in human mitochondrial DNA.

Myoclonus epilepsy with ragged-red fibers (MERRF) has been shown to be associated with a specific point mutation at the nucleotide 8344 in the tRNA(Lys) gene of mitochondrial DNA (mtDNA). We screened 6 patients with clinically diagnosed MERRF and 1 patient with ocular myopathy for point mutations in the tRNA(Lys) gene, using single strand conformation polymorphism (SSCP) analysis, which can detect even a 1-basepair difference between 2 DNA sequences. Using SSCP and consequent DNA sequencing, we identified the known MERRF mutation in 4 out of 6 MERRF patients, as well as in 1 patient with a new clinical phenotype associated with this mutation: progressive external ophthalmoplegia, muscle weakness and a lipoma, but no myoclonus or epilepsy. Two of the patients with clinical MERRF had neither the MERRF-mutation nor any other mutations in the tRNA(Lys) gene. Using SSCP analysis, we also detected a new polymorphism in 1 patient. Thus, SSCP analysis can be applied to search effectively and rapidly for point mutations or polymorphisms in mitochondrial DNA.

Adolescent↗