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PubMed2001

Prediction of DNA single-strand conformation polymorphism: analysis by capillary electrophoresis and computerized DNA modeling.

We have analyzed previously three representative p53 single-point mutations by capillary-electrophoresis single-strand conformation polymorphism (CE-SSCP). In the current study, we compared our CE-SSCP results with the potential secondary structures predicted by an RNA/DNA-folding algorithm with DNA energy rules, used in conjunction with a computer analysis workbench called STRUCTURELAB. Each of these mutations produces measurable shifts in CE migration times relative to wild type. Using computerized folding analysis, each of the mutations was found to have a conformational difference relative to wild type, which accounts for the observed differences in CE migration. Additional properties exhibited in the CE electropherograms were also explained using the computerized analysis. These include the appearance of secondary peaks and the temperature dependence of the electrophoretic patterns. The results yield insight into the mechanism of SSCP and how the conditions of this measurement, especially temperature, may be optimized to improve the sensitivity of the SSCP method. The results may also impact other diagnostic methods, which would benefit by a better understanding of DNA single-strand conformation polymorphisms to optimize conditions for enzymatic cleavage and DNA hybridization reactions.

Base Sequence↗
PubMed1997

Role of myelin basic protein and proteolipid protein genes in multiple sclerosis: single strand conformation polymorphism analysis of the human sequences.

Susceptibility to multiple sclerosis (MS) is widely held to have a strong genetic component. While the identities of genes conferring susceptibility are currently unknown, possible candidates include those genes coding for proteins which function in central nervous system (CNS) myelin. Two such genes are the human myelin basic protein (MBP) and proteolipid protein (PLP) genes, whose products make up approximately 80% of the total protein in CNS myelin. The association of a variable number tandem repeat (VNTR) 5' to the human MBP gene with MS has been the subject of conflicting reports. Here we test the hypothesis that mutations in the human MBP and PLP genes might be associated with MS by examining the entire expressed sequence of both genes by single strand conformation polymorphism (SSCP) analysis, using a panel of 71 MS patients and 71 controls. We have also re-examined the VNTR region in patients and controls. Three base changes were found in the human PLP gene and nine base changes in the human MBP gene; these were essentially equally distributed between patients and controls. No preferential distribution of various alleles of the VNTR between patients and controls was found. Although intronic and regulatory regions have not been examined, it would appear unlikely that mutations in these genes coding for the two major CNS myelin proteins contribute significantly to genetic susceptibility to MS.

Adult↗
PubMed1993

Detection and characterization of new genetic mutations in individuals heterozygous for lactate dehydrogenase-B(H) deficiency using DNA conformation polymorphism analysis and silver staining.

Human lactate dehydrogenase (LDH)--B(H) mutant genes were analyzed by polymerase chain reaction (PCR) and DNA conformation polymorphism. We used polyacrylamide gradient gel and silver staining procedures for DCP analysis, and observed abnormal migration patterns in individuals heterozygous for the LDH-B deficiency. Subsequent sequence determination of the mutant alleles consistently resulted in detection of three single base substitutions (transversions), viz., a C to A at residue "35" (GCG, Ala-->GAG, Glu), a T to G at residue "172" (TTT, Phe-->GTT, Val), and an A to T at residue "176" (ATG, Met-->TTG, Leu). Furthermore, mismatched PCR or amplification refractory mutation system was developed for the rapid screening and confirmation of these mutations. These amino acid replacements may cause conformational changes in neighboring residues; this probably affects the active site arrangement and results in the loss of enzyme activity.

Amino Acid Sequence↗
PubMed1994

Identification of p53 mutations by means of single strand conformation polymorphism analysis in gynaecological tumours: comparison with the results of immunohistochemistry.

The tumour-suppressing gene p53 may undergo mutation by a variety of mechanisms, thus losing its tumour-suppressing activity, and ultimately behaving like an oncogene. The PAb 1801 monoclonal antibody is known to recognise both wild type and mutated p53, although in practice it seems to show a higher reactivity with the mutated gene product in several human tumours. We studied p53 overexpression in a series of 36 human tumours (17 mammary ductal infiltrating carcinomas, 11 endometrial carcinomas and 8 uterine cervical carcinomas) by means of immunohistochemistry using the PAb 1801 antibody and the streptavidin-biotin peroxidase technique. Furthermore, all tumours were screened for mutations in the "hot spot" regions of the p53 gene (exons 5 to 8) by means of SSCP (single strand conformation polymorphism) DNA analysis following amplification of the target exons using the polymerase chain reaction. A good correlation (75-100%) between positive immunohistochemistry and p53 mutations was observed in mammary and endometrial cancer, whereas mutations were detected in only two out of seven immunoreactive cervical carcinomas. Following these results, immunohistochemistry with the PAb monoclonal antibody may be safely used as a screening tool for the detection of mutated p53 in clinical samples of mammary and endometrial cancer, whereas it should be complemented with DNA analysis in cervix carcinoma.

Base Sequence↗
PubMed2000

Catalytic activity of three variants (Ile, Leu, and Thr) at amino acid residue 359 in human CYP2C9 gene and simultaneous detection using single-strand conformation polymorphism analysis.

This study evaluated the catalytic activity of three variants (Ile, Leu, and Thr) at codon 359 of CYP2C9 enzymes expressed in a yeast cDNA expression system, and then established single-strand conformation polymorphism (PCR-SSCP) analysis for simultaneous detection as a screening method. Diclofenac was used for the in vitro experiment, and its hydroxy metabolite (4'-hydroxydiclofenac) was measured by HPLC. To discuss the in vivo effect of the Thr359 variant on the pharmacokinetics of phenytoin, a case report is presented. The efficiency of the SSCP method was evaluated by analyzing DNA samples from a homozygote for Ile359 and a heterozygote for Leu359 or Thr359. To evaluate the interaction between the P450 level and reductase activity, two batches of the Thr359 variant with a different P450:reductase activity ratio (1:4.0 and 1:1.4) were used. The in vitro study revealed that recombinant Ile359, Leu359, and Thr359 (2 batches) possessed a mean Km of 2.0, 16.5 and (3.8 and 2.9) micromol and Vmax of 12.4, 17.9 and (4.4 and 5.1) nmol/min/nmol P450, respectively. Although the magnitude of the change in catalytic efficiency for the Thr359 variant was close to that of the Leu359 variant, the effect of the two variants on diclofenac 4'-hydroxylation appears to be different because Leu359 variant was associated with a high Km, and Thr359 with a low Vmax. No significant differences in the kinetic data were observed between the two Thr359 enzymes, suggesting that low reductase activity in the Thr359 enzyme was not a major determinant in the present in vitro experiment. Estimated pharmacokinetic parameters of phenytoin obtained by the Bayesian method in an epileptic patient who was a heterozygote carrier for Thr359 variant were: Km = 6.45 microg/mL, Vmax = 5.77 mg/kg/d, and Vmax/Km = 0.89 L/kg/day. The Vmax/Km value in this patient was similar to the population mean value (0.90 L/kg/day) in Japanese heterozygotes for the Leu359 variant. Results for PCR-SSCP were in complete agreement with those obtained using established methods. Thus, the PCR-SSCP approach is useful for identifying these three variants of the CYP2C9 gene.

Adult↗
PubMed1997

Determination of the genome composition of influenza virus reassortants using multiplex reverse transcription-polymerase chain reaction followed by fluorescent single-strand conformation polymorphism analysis.

The influenza virus genome is composed of eight negative-strand RNA segments. In cells coinfected with two or more influenza strains, the genomic RNAs reassort at random, resulting in progeny viruses (reassortants) that contain genes derived from each parent. Genetic reassortment among influenza viruses occurs naturally and plays an important role in viral epidemiology and pathogenicity. The reassortment process is also utilized for the annual production of influenza vaccines. Each year, the two gene segments that encode the major surface antigens of the current virulent, wild-type viruses are reassorted with the remaining six gene segments of a laboratory-derived vaccine or "master donor" strain. As the gene reassortment appears to be random, identifying a progeny virus with the desired gene constellation can be labor-intensive. We developed a streamlined, cost-effective method to genotype influenza viruses that combines multiplex reverse transcription-polymerase chain reaction (RT-PCR) and fluorescent single-strand conformation polymorphism (SSCP) analysis. This method utilizes oligonucleotide primers labeled with one of three fluorescent dyes to generate RT-PCR products for each gene segment in a multiplex configuration. The RT-PCR products of the reassortants, wild-type, and master donor viruses are then electrophoresed under SSCP conditions. The viral origin of each gene segment can be identified by fluorescence and mobility shift patterns of the corresponding RT-PCR products. We demonstrate the utility of this method in differentiating the genes of a master donor strain, several wild-type viruses, and vaccine reassortants.

Animals↗
PubMed1993

DNA conformation polymorphism analysis of DR52 associated HLA-DR antigens by polymerase chain reaction: a simple, economical and rapid examination for HLA matching in transplantation.

HLA-DRB1 and -DRB3 alleles of DR52-associated (DR52ass) HLA-DR antigens were genotyped by a polymerase chain reaction (PCR) - based simple and practical method. Genomic DNAs from two hundred Japanese panels were subjected to PCR with two pairs of primers to separately amplify the DR52ass-DRB1 (DR3, 5, 6, and 8) alleles and DRB3 (DR52) alleles. The specific amplification revealed that 128 and 76 panels possessed DR52ass alleles and DRB3 alleles, respectively. PCR products from these panels were heat-denatured, electrophoresed in a non-denaturing polyacrylamide gel, and visualized by silver staining. Electrophoretic mobilities of the DNA samples were compared with those of the typing standards with known genotypes of DR52ass-DRB1 and DRB3 alleles. This method, designated PCR-DNA conformation polymorphism (DCP) analysis, allowed genotyping of the DR52ass-DRB1 and DRB3 alleles of panels without any sequence-specific oligonucleotide probe (SSOP) or restriction endonuclease, and the entire process after PCR could be completed within a few hours. Because the DR52ass-DRB1 and DRB3 alleles assigned by this method were shown to be identical to those determined by the PCR-SSOP method, PCR-DCP analysis was suggested to be a simple and practical HLA genotyping method.

Alleles↗
PubMed1995

Single-strand conformation polymorphism analysis by perpendicular temperature-gradient gel electrophoresis.

Using a newly developed temperature-gradient gel electrophoresis apparatus, mutations in the K-ras oncogene and p53 tumor suppressor gene were analyzed for single-strand conformation polymorphism (SSCP). The mobilities of single-stranded DNAs, carrying various mutations, change--depending on the gel temperature during electrophoresis. Therefore, temperature-gradient single-strand conformation polymorphism (TG-SSCP) analysis can provide useful information concerning the optimum temperature for SSCP and may also be used to screen for various mutations or polymorphisms in a single electrophoretic run.

Base Sequence↗
PubMed2003

Analysis of conformational equilibria in aplidine using selective excitation 2D NMR spectroscopy and molecular mechanics/dynamics calculations.

Aplidine (dehydrodidemnin B), a natural product with potent antitumor activity currently in multicenter phase II clinical trials, exists in DMSO as a mixture of four slowly interconverting conformations in a ratio of 47:33:13:7. NMR spectroscopy shows that these arise as a consequence of cis/trans isomerization about the NMe-Leu(7)-Pro(8) and Pro(8)-Pyr amide bonds of the molecule's side chain. Two major conformations account for 47% and 33% of the total population, a ratio of 60:40 between the two. They correspond to the cis- and trans-isomers, respectively, about the Pro(8)-Pyr amide bond. Two minor conformers arise as a consequence of similar isomerism about the Pro(8)-Pyr amide bond, but in structures in which the NMe-Leu(7)-Pro(8) amide bond is cis rather than trans. These account for approximately 13% and 7% of the total population, corresponding to a ratio of 65:35 cis/trans, respectively. Molecular dynamics simulations show that the three-dimensional structures of all four conformational isomers are similar in the macrocycle and that all are essentially unchanged with respect to the macrocycle of didemnin B. Significant differences in the conformation of the molecule's side chain are, however, observed between major and minor pairs. Analysis of hydrogen-bonding patterns shows that each major conformer exhibits a beta-turn like structure and is stabilized by hydrogen bonding between a different carbonyl group of the pyruvyl unit of the molecule's side chain and the NH of the Thr(6) residue. The minor isomers have a cis-amide bond between the NMe-Leu(7) and Pro(8) residues that obliges the side chain to adopt an extended disposition where hydrogen bonding to the macrocycle is absent. These results suggest that the ability of the molecule's side chain to adopt a beta-turn-like conformation may not be a prerequisite for biological activity in the didemnins and that conformations having an extended side-chain may play a role in the biological activity of aplidine.

Amides↗
PubMed1996

Single-strand conformation polymorphism analysis of PCR-amplified ribosomal DNA internal transcribed spacers to differentiate species of Aspergillus section Flavi.

A novel genetic approach for classifying the species of Aspergillus Section Flavi is described here. This approach consists of PCR amplification of the 5.8S ribosomal DNA-intervening internal transcribed spacer regions (ITS I-5.8S-ITS II) with universal primers and of analysis of the PCR product by the principle of single-strand conformation polymorphism (SSCP). The approximately 570- to 590-bp PCR products were denatured and subjected to electrophoresis on a polyacrylamide gel supplemented with 20% formamide. The SSCP patterns of these species became more distinct by the addition of formamide to the gel and by visualization with ethidium bromide staining. A little interspecific length polymorphism among amplified ribosomal DNAs was enhanced to be detected by PCR-SSCP analysis. This analysis was capable of classifying 67 of the 68 Aspergillus Section Flavi strains tested into the following four groups, regardless of origin: A. flavus/A. oryzae, A. parasiticus/A. sojae, A. tamarii, and A. nomius. The results of restriction fragment length polymorphism analysis with PCR products of the ITS regions were consistent with those of PCR-SSCP analysis, except for A. nomius, which was not clearly differentiated from A. parasiticus/A. sojae. Nonradiolabelled PCR-SSCP analysis is inexpensive and practical to perform without special apparatus or skill and should assist in fungal morphological identification.

Aspergillus↗
PubMed1995

Detection of p53 gene mutations in aspiration biopsy specimens from suspected breast cancers by polymerase chain reaction-single strand conformation polymorphism analysis.

Genomic DNA was extracted from aspiration biopsy specimens taken from 15 suspected cases of breast cancer, including 7 known cases of breast cancer, and the p53 gene was studied for evidence of mutation by using a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. In 5 of the 15 cases (33%), p53 gene mutation was identified and these tumors were subsequently histologically diagnosed as malignant. Further, DNA flow cytometry of the 15 tumors demonstrated that 6 (40%) were aneuploid and malignant, whereas 9 (60%) were diploid and benign. It was also found that the tumor cells in 5 aspirated cases that showed p53 gene mutations were all aneuploid, the p53 protein expression was positive, and the tumors were proved to be histologically malignant. It was thus concluded that the detection of p53 gene mutation by PCR-SSCP analysis of aspirated biopsy specimens from suspected breast cancers is a helpful method for achieving a more accurate diagnosis.

Adult↗
PubMed2001

Genotyping Cryptosporidium parvum by single-strand conformation polymorphism analysis of ribosomal and heat shock gene regions.

Polymerase chain reaction (PCR)-coupled single-strand conformation polymorphism (SSCP) approaches utilizing nuclear DNA regions of the small subunit (SSU) of ribosomal RNA and heat shock protein 70 gene (HSP70) were established for genotyping Cryptosporidium parvum. The regions were amplified (individually or in a multiplex reaction) by PCR from DNA extracted from oocysts from ruminant or human hosts, then denatured and subjected to electrophoresis in a mutation detection enhancement (nondenaturing) gel matrix. Single-strand profiles produced in SSCP allowed the unequivocal identification/differentiation of the two common (human, 1 and cattle, 2) genotypes of C. parvum and the direct display of sequence variability within some samples, reflecting population variation. As these are considered among the most closely related genotypes (based on SSU and HSP70 sequence data), these findings and other preliminary results for C. felis (from cat) C. serpentis (from snake) and C. baileyi (from bird) indicate that the SSCP approaches established could be employed to identify any of the currently recognised genotypes and species of Cryptosporidium.

Animals↗
PubMed1989

Conformational energy analysis of the pentapeptide Ac-Arg-Asn-Cys-Tyr-Asn-NMA from alpha 1-purothionin.

Conformational energy analyses were carried out on the pentapeptide RNCYN (Ac-Arg-Asn-Cys-Tyr-Asn-NMA) and on related peptides. RNCYN is a highly conserved amino acid sequence in thionins and viscotoxins. The conformation of the pentapeptide was calculated to be an amphipathic alpha-helix, with the tyrosine and cysteine on the nonpolar side of the helix and the arginine and both asparagines on the polar side. Our results are inconsistent with the conformation determined using the Chou-Fasman prediction method, but are consistent with the conformation determined experimentally (using n.m.r.) for this pentapeptide sequence in alpha 1-purothionin.

Amino Acid Sequence↗
PubMed1996

Fluorescence-based polymerase chain reaction-single-strand conformation polymorphism analysis of p53 gene by capillary electrophoresis.

Mutation of the p53 gene plays an important role in neoplastic progression in human tumorigenesis. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) techniques are now available for the detection of point mutations. The original method using polyacrylamide gel electrophoresis is disadvantageous, particularly for clinical tests and for analysis of large numbers of samples. Therefore, using an automated capillary electrophoresis (CE) technique with a molecular-sieving polymer solution, we have devised a completely automatic fluorescence-based PCR-SSCP system (CE-FSSCP) for the differential detection of point mutations that dose not require SSCP with radioisotopes and polyacrylamide gels. The automatic CE-FSSCP system was developed for reproducible operations in the denaturation of double-stranded DNA and electrophoresis of single-stranded DNA. The detection system consists of a 100 W I2 lamp and photomultiplier. We performed CE-FSSCP with a 2% linear polyacrylamide polymer solution containing 5% glycerol. Four tissue specimens of lung tumors with mutations in exon 7 of the p53 gene were found to have mutant alleles; six-base-pair deletion at codons 247-248, a one-base-pair deletion at codon 260, a one-base-pair deletion at codon 244 and a GGC to CGC substitution at codon 244. We expect this technique to prove useful for the clinical DNA diagnosis of human cancers, determination of the therapeutic effect of anticancer agents and for the study of the molecular aspects of the mechanisms involved in the pathogenesis of human cancers.

Alleles↗
PubMed1992

p53 gene mutations in esophageal cancer detected by polymerase chain reaction single-strand conformation polymorphism analysis.

Mutations of the p53 gene play an important role in the development of common human malignancies. We investigated mutations of this gene in 26 surgical specimens of esophageal cancer using the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis. The results were correlated with histological findings, DNA ploidy and the short-term relapse of the disease. PCR-SSCP analysis detected mutations of the p53 gene in 10 tumors (38%), eight in exons 5-6 and two in exons 7-8. A higher incidence of lymph node metastasis, poorly differentiated tumor, DNA aneuploidy and short-term relapse of the disease was observed in cases with p53 gene mutations, although the findings were not statistically significant.

Base Sequence↗
PubMed1992

Detection of point mutations in human DNA by analysis of RNA conformation polymorphism(s).

RNA molecules were found to separate into numerous metastable conformational forms upon non-denaturing gel electrophoresis. The equilibration of the conformations was accelerated by heating or mild denaturing conditions. Single-base substitutions in the sequence of the RNAs caused changes in the conformational patterns, including mobility shifts of major and minor conformations, appearance of new conformations and loss of other conformations. This sequence-dependent RNA conformational polymorphism was used to detect point mutations in p53 and, dihydrofolate reductase genes. Sense and anti-sense RNA strands corresponding to the same segment of the p53 gene gave entirely different conformational patterns. To generate the RNA, short regions of the target genes (up to about 250 bp) were amplified by the polymerase chain reaction and the resulting DNA segments transcribed to RNA by T7 RNA polymerase. The method is rapid, simple, amenable to non-radioactive visualization and was successful in several cases when DNA single-strand conformational polymorphism analysis (Orita et al. (1989) Genomics 5, 874-879) failed to detect the point mutation.

Base Sequence↗
PubMed1999

The procedure of polymerase chain reaction-restriction fragment-single strand conformation polymorphism analysis by Hha I/Hinc II to detect mitochondrial DNA mutations.

We reported 56 mitochondrial DNA (mtDNA) mutations in Japanese compared with the Cambridge Sequence by polymerase chain reaction-restriction fragment-single strand conformation polymorphism (PCR-RF-SSCP) analysis. Here we report the principle and the detailed procedures of PCR-RF-SSCP analysis. Restriction map of the 15,673 bp PCR product of mtDNA was designed by MacMolly Tetra, and Hha I and Hinc II were selected. Restriction fragment length polymorphism (RFLP) analysis of 15,673 bp PCR products by Hha I/Hinc II revealed one common pattern and 10 polymorphic patterns compared with the prediction based on the Cambridge Sequence. When the samples were electrophoresed by SSCP analysis, DNA fragments of 912 bp, 772 bp, 761 bp, 634 bp, 431 bp, 327 bp, 226 bp and 171 bp showed 6, 4, 5, 4, 5, 7, 2 and 3 polymorphic patterns, respectively. The determinations of nucleotide sequence of these 46 polymorphic patterns revealed 56 mutations compared with the Cambridge Sequence and 7 of these mutations were common in Japanese. Among these, 20 mutations have not been reported before.

DNA, Mitochondrial↗