[Analysis of conformation of oligonucleotide with sequences 5'-d(GpCpGpApApGpC) by 1H-NMR-spectroscopy].
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Animals↗
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Complete subtyping of the HLA-A locus by sequence-specific amplification followed by direct sequencing or single-strand conformation polymorphism analysis.
A variety of reasons related to the HLA class I system has complicated the application of molecular approaches to HLA class I typing. Here we present a PCR-based HLA-A typing strategy considering the sequence variations of the two most polymorphic exons which allows complete subtyping of the HLA-A locus. The method is based on a sequence-specific amplification identifying the serologically defined HLA-A specificities. The PCR products generated by these group-specific primers bear the sequence information necessary for a postamplification specificity step. The primer pairs are located within one exon, either exon 2 or exon 3, which avoids amplification of polymorphic intron sequences allowing subsequent single-strand conformation polymorphism analysis and facilitating direct sequencing. Using this method we investigated 48 cell lines and 153 clinical samples. 23 PCR reactions are performed per individual for the assignment of the serological specificities A1-A80. The reproducibility was 100% in all cell lines and 85 clinical samples typed on two separate occasions. With the exception of 13 out of 231 possible serological combinations all homozygous and heterozygous combinations of A1-A80 can be distinguished by specific amplification patterns. Comparing the PCR based typing results with those of serology in 12% a discrepancy was found. Solid-phase sequencing or SSCP analysis of the group-specific PCR fragments allowed complete subtyping of the HLA-A locus. This strategy can identify all 48 HLA-A alleles based on the sequence variations of the 2nd and 3rd exon. 1128 homozygous and heterozygous allele combinations are possible for the HLA-A locus. Only 4 out of these 1128 allele combinations remained unresolved.
Alleles↗
High resolution for single-strand conformation polymorphism analysis by capillary electrophoresis.
Since the successful completion of the Human Genome Project, increasing concern is being directed toward the polymorphic aspect of the genome and its clinical relevance. A form of single-strand DNA-conformation polymorphism analysis (SSCP) employing nondenaturing slab-gel electrophoresis (SGE) is applicable to the genetic diagnosis of bladder cancer from urine samples. To bring this technique into routine clinical practice, the use of capillary electrophoresis (CE) is naturally favorable in terms of speed and automation. However, the resolving power of SSCP, a prerequisite basis for reliability required in diagnostics, remains as a challenge for CE systems. We thus focused on this topic and conducted studies on CE instruments equipped with a single capillary or an array of multiple capillaries, using the resolution (Rs) as a quantitative scale for the resolving power. Polymer concentration and buffer are shown to be the decisive parameters. High Rs values of >2.5 are achieved for representative SNPs markers under the optimized conditions, without sacrificing such intrinsic advantages of CE over SGE as the 10-fold quicker migration time and operation that is reproducible, continuous, and automatic. The strategies presented broaden the limits of CE in both the current and related applications.
Buffers↗
Detection of mutations and polymorphisms in the p53 tumor suppressor gene by single-strand conformation polymorphism analysis.
Deciphering the genetic mechanisms in cancer development requires analysis of a large number of tumors for consistent genetic alterations. Single-strand conformational polymorphism (SSCP) analysis is a fast and efficient method for detecting mutations, deletions, insertions and loss of alleles. The primary advantage of this method is speed and ability to screen a large number of samples at one time. Here we report the use of the SSCP technique for rapidly screening tumor and normal tissues for mutations and polymorphisms in the p53 tumor suppressor gene. Because the DNA extracted from specific aberrant bands from different samples always give rise to the same nucleotide sequence upon sequencing analysis, the SSCP technique can be used as a diagnostic tool to identify the presence of such genetic alterations without having to spend time on further sequencing analysis.
Genes, p53↗
Rapid and sensitive genotyping of Epstein-Barr virus using single-strand conformation polymorphism analysis of polymerase chain reaction products.
Two distinct wild-type Epstein-Barr virus (EBV) strains (A and B) that have significantly diverged at the two small RNA-encoding region (EBER) have been identified (Arrand et al., 1989). In order to test whether single-strand conformation polymorphism analysis (SSCP) would correlate with these sequence variations, we designed primer pairs specific for EBER-encoding regions for amplification of divergent sequences by polymerase chain reaction (PCR). The PCR-amplified products from six EBV-positive cell lines were analyzed by SSCP method, and the results were compared with the prototype strains B95-8 (type A) and AG876 (type B). Type-specific point mutations were detected as demonstrated by shifts in mobility due to conformational changes of DNA sequences. The locations of point mutations were identified by direct sequencing of the PCR amplified DNA. Of the three primer pairs designed, the pair that amplified a 190 bp fragment spanning six type-specific point mutations gave the best resolution in SSCP analysis. This pair is now preferred for initial genotyping of EBV-infected tumor biopsies. Thus, SSCP is a simple, fast and efficient technique for genotyping of EBV-associated diseases.
Antigens, Viral↗
An efficient strategy for detection of known and new mutations of the CYP2D6 gene using single strand conformation polymorphism analysis.
To detect mutations in the cytochrome P450 CYP2D6 gene (CYP2D6), we developed a strategy based on single-strand conformation polymorphism (SSCP) analysis of the gene amplified by polymerase chain reaction (PCR). The efficiency of the method was evaluated by analysing DNA samples from extensive metabolizers (EM) and poor metabolizers (PM) of debrisoquine. Haplotypes, alleles and mutations of CYP2D6 had previously been characterized in each individual using PCR assays, Xba I restriction fragment length polymorphism (RFLP) and sequencing. PCR-SSCP results were in complete agreement with those obtained using established methods. All previously characterized mutations were associated with particular shifts in the electrophoretic mobility of DNA fragments allowing their identification. We further tested the efficiency of PCR-SSCP for detecting new CYP2D6 mutations. DNA from a PM subject presumed to carry an unknown non-functional mutant allele of CYP2D6 was amplified and bands with aberrant migration patterns were observed on SSCP gels. Sequence analysis of the corresponding DNA fragments revealed the causative mutations. In this way, a novel non-functional allele of the gene, carrying three previously reported mutations and a new mutation in the third exon which results in a premature termination codon, was characterized. Finally, CYP2D6 SSCP analysis was performed on DNA amplified with fluorescent primers and an automated DNA sequencer was used for SSCP analysis of products. We conclude that the PCR-SSCP approach is a powerful method of identifying simultaneously known and new mutations of the CYP2D6 gene.
Alleles↗
Multivariate curve resolution: a powerful tool for the analysis of conformational transitions in nucleic acids.
A successful application is reported of the multivariate curve resolution alternating least-squares method (MCR-ALS) for the analysis of nucleic acid melting and salt-induced transitions. Under conditions where several structures co-exist in a conformational equilibrium, MCR-ALS analysis of the UV and circular dichroism (CD) spectra at different temperatures, ionic strength and oligonucleotide concentration allows for the resolution of concentration profiles and pure spectra of the different species. The methodology is illustrated by the case of the cyclic oligonucleotide d . The melting transition of this molecule at different oligonucleotide concentrations was studied at 0, 2 and 10 mM MgCl2 by UV and CD spectroscopy. In addition, salt titration experiments were carried out at 21.0 and 54.0 degrees C. The MCR-ALS analysis indicates that three different conformations of this molecule co-exist in solution. In agreement with previous NMR studies, these conformations were assigned to a monomeric dumbbell-like structure, a dimeric four-stranded conformation and a disordered (random coil) structure. The MCR-ALS methodology allows for a detailed analysis of how this equilibrium is affected by temperature, salt and oligonucleotide concentration.
Algorithms↗
[Rapid detection of platelet glycoprotein IIb, IIIA gene variety by single strand conformation polymorphism analysis].
Glanzmann thrombasthenia (GT) is the most common inherited disorder of platelet defective function. Most of the molecular defects of GT identified in recent years have been caused by point mutation in the platelet glycoprotein (GP) IIb, IIIa genes. Trying to rapidly detect point mutation (or subtle variety) in GPIIb, IIIa genes, we have used single strand conformation polymorphism analysis (SSCPA) and studied the gene structure of platelet GPIIb. IIIa from normal controls and patients with Glanzmann thrombasthenia. After DNA preparation from peripheral blood, all the exons of GPI-Ib, IIIa were amplified by PCR. Denaturated PCR products were separated on mini polyacrylamide gel and stained by silver staining method in the Phast System. Only the PCR products with different migration on the gel were sequenced. 2 GT patients with point mutations and 1 GT patient with 16 bp deletion were found in our study. At the same time, we determined 3 polymorphisms in GPIIb gene. We conclude that SSCPA is a rapid, sensitive and effective method for detecting platelet IIb, IIIa gene variety.
DNA Mutational Analysis↗
[Mutation of p53 gene in the transformed fibroblasts detected by nonradioisotopic single strand conformation polymorphism analysis].
The human embryonic lung fibroblasts transformation was induced by chemical mutant Glycidyl Methacrylate (GMA) in vitro. Transformed clonies were isolated and then exon 5 and exon 8 of p53 gene were specifically amplified by polymerase chain reaction. Amplified fragments were detected by using silver stained single strand conformation polymorphism analysis. The results showed that exon 8 altered in the transformed cells, indicating that p53 gene played an important role in the human embryonic lung fibroblasts transformation induced by GMA.
Air Pollutants, Occupational↗
Use of TP53 reference materials to validate mutations in clinical tissue specimens by single-strand conformational polymorphism analysis.
BACKGROUND: As genetic information moves from basic research laboratories in to the clinical testing environment, there is a critical need for reliable reference materials for the quality assurance of genetic tests. A panel of 12 plasmid clones containing wild-type or point mutations within exons 5-9 have been developed as reference materials for the detection of TP53 mutations. AIM: The goal of this study was to validate the reference materials in providing quality assurance for the detection of TP53 mutations in clinical specimens. METHODS: We studied 33 gynecological samples, 11 apparently normal samples and 22 malignant tumors of various origins. Mutations were identified using single-strand conformational polymorphism analysis with both slab gel and capillary electrophoresis. All DNA samples were amplified with fluorescently labeled PCR primers specific for exons 5-9 for mutation detection. RESULTS: Of the 33 patient samples tested, mutations and polymorphisms were found in six specimens in three of the five exons scanned; no mutations were found in exons 7 or 9. Both a mutation and polymorphism were found in non-malignant specimens from the control group. The mutations were confirmed by DNA sequence analysis of the regions scanned. CONCLUSIONS: Mutations and polymorphisms were detected in the clinical samples. All of the mutations were silent except for one non-conservative mutation in exon 5, codon 181. This study demonstrates the usefulness of the National Institute of Standards and Technology (NIST) TP53 reference panel in TP53 mutation detection in clinical tissue specimens.
Adolescent↗
Detection of single nucleotide polymorphisms in coagulation factor XI deficient patients by multitemperature single-strand conformation polymorphism analysis.
Factor XI (FXI) deficiency is a rare inherited disorder which can cause bleeding complications especially in case of hemostatic challenge and/or in tissues with high fibrinolytic activity. A number of causative mutations have been described in FXI deficient individuals which have been detected by various screening methods. In this study, we present the application of the multitemperature single-strand conformation polymorphism analysis (MSSCP) on the FXI gene, a recently developed methodology for the detection of single nucleotide exchanges. We analyzed a total of 217 polymerase chain reaction (PCR) fragments from the promoter region as well as from exons 1-7 and 11-15 and compared the results to automatic fluorescent sequencing. A total of 29 PCR fragments showed single nucleotide exchanges in conventional fluorescent sequencing, representing 10 different mutations (nine missense mutations, one small deletion) and four frequent polymorphisms. With MSSCP electrophoresis at a standard temperature profile (gel temperature 35-20-10 degrees C) we were able to detect 13 of 14 (93%) different nucleotide exchanges in 25 of 29 PCR fragments (86%). Hence, the detection rate for genetic variations in the FXI gene was 86%. To evaluate the reproducibility, MSSCP was performed twice for 174 PCR fragments and the consistency between two electrophoretic runs was 99%. We conclude that the MSSCP is a sensitive, fast, and cost effective screening method for the detection of FXI gene mutations.
DNA Mutational Analysis↗
[SSCP (single strand conformation polymorphism) analysis for detection of point mutations. A technique and its limits exemplified by dominantly inherited forms of diabetes (MODY)].
BACKGROUND: Molecular biological techniques of mutation detection become more and more a focus of interest in clinical medicine because the responsible genes are now known for an increasing number of diseases and the detection is essential to enable reliable predictions and to design individual therapies. Therefore the SSCP (Single Strand Conformation Polymorphism) analysis is one of the most commonly applied methods in detection of point mutations. SSCP ANALYSIS: The use of SSCP for detection of the pathogenic mutations of MODY (Maturity Onset Diabetes in the Young) is described as an example. Handling, efficiency and costs are critically analyzed. In a blind test, this method was only partly suitable for reliable analysis. Particularly diseases without a distinct hotspot and with a widespread distribution pattern of mutations throughout the coding sequence require sequencing as the gold standard.
DNA Mutational Analysis↗
Molecular study of eight Japanese cases of glucose-6-phosphate dehydrogenase deficiency by nonradioisotopic single-strand conformation polymorphism analysis.
Using a newly developed nonradioisotopic method of polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis combined with the direct sequencing using the fluorescence-labeled terminator, we identified seven missense mutations, 527 A-->G, 1003 G-->A, 1159 C-->T, 1160 G-->A, 1229 G-->A, 1246 G-->A, and 1361 G-->A, in eight Japanese patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Except for the 527 A-->G, each mutation has been reported to cause variants G6PD Chatham, G6PD Guadalajara, G6PD Beverly Hills, G6PD "Japan", G6PD Tokyo, and G6PD Andalus, respectively. In addition, a single base deletion in intron 5 was found in the patients with G6PD Guadalajara or G6PD Andalus. The variant with unique 527 A-->G was characterized and designated as G6PD Shinshu. We also characterized G6PD "Japan" and found that the variant had the striking resemblance with G6PD Riverside, bearing a missense mutation in the same codon, but causing a different amino acid substitution. Our modified PCR-SSCP analysis using minigel and ethidium bromide staining could detect six of the eight diverse mutations in the G6PD gene. Because it is easy and requires no special apparatus, this modified method will be useful for screening mutations in the G6PD gene.
Base Sequence↗
Direct, automated detection of rifampin-resistant Mycobacterium tuberculosis by polymerase chain reaction and single-strand conformation polymorphism analysis.
A rapid screening test was recently established for the detection of mutations in the rpoB gene of Mycobacterium tuberculosis, a region identified as the locus for rifampin resistance (Rifr). The detection method involved the amplification by polymerase chain reaction (PCR) of the Rifr region and the identification of mutations by single-strand DNA conformation polymorphism analysis (SSCP) of the amplification products. Experience using two different PCR-SSCP formats for the evaluation of BACTEC cultures and sputum is presented here; the previously described manual procedure for the detection for the detection of radiolabelled amplification products and an automated SSCP by which fluorescein-labelled products were detected on a Pharmacia DNA sequencer apparatus. All 17 different Rifr mutations known to date were consistently detected. PCR-SSCP could be used for the evaluation of minimally grown cultures (BACTEC 12B medium with a growth index of < or = 100) and for direct screening of microscopically positive sputa with greater than 10 organisms per field (magnification, x250). Implementation of this technique could result in rapid detection of rifampin resistance in M. tuberculosis, a marker of multidrug-resistant tuberculosis.
Base Sequence↗
Cathepsin D: screening for new polymorphisms using single-strand conformation polymorphism analysis.
Cathepsin D (CTSD) is a lysosomal protease involved in the pathogenesis of several diseases such as breast cancer and possibly Alzheimer's disease (AD). Previous findings revealed a significant association between the T allele of the 224 C/T (A58V) polymorphism in exon 2 of the CTSD gene and late onset AD. The exonic regions of the CTSD gene were screened for further polymorphic variations using polymerase chain reaction and single-strand conformation polymorphism analysis. In addition to the known 224 C/T polymorphism and two silent mutations in exons 3 and 4 we detected two new polymorphisms in introns 5 and 8. Combination of these sequence variations results in three different haplotypes; one of these haplotypes is due to the new polymorphism in intron 5. We detected no further missense mutations except for the known 224 C/T polymorphism in exon 2. Thus, if sequence variations within the CTSD gene influence the risk for various diseases, the pathogenic mechanism is likely to be linked to the amino acid substitution in the profragment of CTSD.
Adult↗
Metastable single-strand DNA conformational polymorphism analysis results in enhanced polymorphism detection.
Single-strand DNA conformational polymorphism (SSCP) makes use of sequence-dependent folding of single-stranded DNA (ssDNA), which alters the electrophoretic mobility of the fragments, to detect sequence differences between closely related molecules. In this study ssDNAs were purified by depletion of the complementary strand and PCR reactants on magnetic M-280-strepavidin beads. It was found that SSCP profiles created by purified ssDNAs differ from the profiles created by more usual SSCP methods. Under some conditions, SSCP profiles using whole PCR reaction products may result from the interaction between residual PCR primers and ssDNAs. We observed that the ratio of conformers revealed by band position and band intensity may vary between the assay techniques and misinterpretation of sequence variants may result. Another observation of this study was the formation of metastable conformational isomers with bead-purified ssDNAs by eliminating the thermal treatment used in conventional SSCP methods. The metastable SSCP (mSSCP) represents a novel and sensitive system for detection of sequence variation between closely related DNAs. The technique used here for the preparation of the purified ssDNAs is potentially useful for automated PCR-SSCP analysis using capillary electrophoresis or other methods.
Autoradiography↗
Single-strand conformation polymorphism analysis coupled with stratified DNA sequencing reveals reduced sequence variation in the su(s) and su(wa) regions of the Drosophila melanogaster X chromosome.
Single-strand conformation polymorphism (SSCP) analysis followed by DNA sequencing of stratified sub-samples was used to survey DNA polymorphism in the su(s) and su(wa) regions in a natural population of Drosophila melanogaster. su(s) and su(wa) are located near the telomere of the X chromosome, where the rate of crossing over per kilobase of DNA monotonically decreases toward the tip. SSCP was assessed in 12 noncoding segments amplified from the su(s) region (3213 bp) and in 8 noncoding segments amplified from the su(wa) region (1955 bp). Sets of segments were multiplexed in a single electrophoretic lane to increase the number of base pairs assayed per lane. Eight segments were monomorphic, and the other 12 segments exhibited two to four SSCP classes. Only four within-SSCP-class DNA sequence differences (a single nucleotide substitution) were observed among 24,360 bp compared within classes. The between-SSCP-class DNA sequence comparisons revealed 27 substitutions and 9 insertion/deletion polymorphisms. The average numbers of substitutional differences per site were 0.0010 and 0.0021 for su(s) and su(wa), respectively. These values are intermediate between those reported for the more distal y-ASC region (0.0004) and the more proximal Pgd locus (0.0024). This observation is consistent with the prediction of the hitchhiking-effect model-i.e., a monotonic increase in polymorphism as a function of crossing over per kilobase.
Animals↗
Effect of polymer matrix and glycerol on rapid single-strand conformation polymorphism analysis by capillary and microchip electrophoresis for detection of mutations in K-ras gene.
We present the rapid single-strand conformation polymorphism (SSCP) analysis by capillary and microchip electrophoresis to detect the mutations in K-ras gene. Parameters that might affect the analysis of mutation in K-ras gene, such as the polymer and the additive in the sieving matrix, have been studied systematically. Under the optimal conditions, the analysis of seven mutants of K-ras gene could be finished within 10 min by capillary electrophoresis (CE). Furthermore, with the wild-type gene as the inner standard, the analysis accuracy of mutations could be improved. In addition, by studying the properties of polymer solutions, the matrix suitable for microchip electrophoresis was found, and the detection of mutations in K-ras gene could be further shortened to 1 min.
Base Sequence↗