Single-strand Conformation Polymorphism DNA Research Articles

PCR-SSCP Analysis of the Ribosomal DNA ITS Regions of the Willow Rust Fungi in Japan.

Single-strand DNA conformation polymorphism (SSCP) analysis using internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) amplified by polymerase chain reaction (PCR) was performed to detect DNA polymorphism of nine species of Melampsora rust fungi on willows. The rDNA ITS 1 region (approximately 300-320bp in size), was amplified from 49 collections. The SSCP patterns in the amplified ITS 1 region of five species, M. capraearum, M. epiphylla, M. larici-urbaniana, M. microsora and M. yezoensis were species-specific. On the other hand, no distinction was found in the SSCP patterns between M. chelidonii-pierotii and M. coleosporioides, and between M. epitea and M. humilis. In improved PCR-SSCP analysis using restriction fragments of the amplified ITS 1 and ITS 2 regions, no difference was detected between M. chelidonii-pierotii and M. coleosporioides, and between M. epitea and M. humilis. These results suggest the respective two species have a close genetic and taxonomical relationship. We revealed the PCR-SSCP analysis may be beneficial as a tool for distinguishing species of Melampsora because the analysis is simple and sensitive.

Assessment of mutations in Ki-ras and p53 in colon cancers from azoxymethane- and dimethylhydrazine-treated rats.

Mutations in the Ki-ras oncogene and the p53 tumor suppressor gene are known to occur at high frequencies in human colon cancers. We measured the frequency of mutations in these two genes in colon adenocarcinomas obtained from a widely used experimental model of human colon carcinogenesis: F344 rats treated with the carcinogens azoxymethane (AOM) or dimethylhydrazine (DMH). We detected codon 12 mutations in Ki-ras in approximately 60% of colon adenocarcinomas induced by either carcinogen. We characterized the rat p53 intron-exon junctions to construct primers for polymerase chain reaction amplification of this gene. We discovered that the rat p53 gene was structurally different from the human p53 gene, as the rat gene was missing one intron between exons 6 and 7. Both single-stranded DNA conformational polymorphism analysis and direct DNA sequencing of the highly conserved regions of rat exons 5-7 were conducted because the corresponding human regions (exons 5-8) have been reported as being mutated most frequently in human colon cancers. Using these methods, we were unable to identify any p53 mutations in the highly conserved regions of exons 5-7 in either AOM- or DMH-induced colon adenocarcinomas. These data confirm that Ki-ras was mutated in most colon cancers in AOM- or DMH-treated rats but indicate that molecular alterations in the p53 gene, if they occur in this animal model, are different from most p53 mutations in human colon cancers.

Characterisation of CAH alleles with non-radioactive DNA single strand conformation polymorphism analysis of the CYP21 gene.

The major cause of congenital adrenal hyperplasia (CAH), a common recessive genetic disease, is the deficiency of steroid 21-hydroxylase (21OH), a microsomal enzyme encoded by the CYP21 gene. Although several...

DNA diagnosis of X-linked amelogenesis imperfecta (AIH1).

Mutations in the amelogenin gene, AMGX, are known to cause X-linked amelogenesis imperfecta (AIH1). We have used DNA single-strand conformational polymorphism analysis and DNA sequencing to diagnose this disorder unequivocally in two related boys aged 3 and 7 years, respectively, from a family in which an existing mutation in the amelogenin gene is segregating.

Polymorphisms of the TAP1 and TAP2 transporter genes in Japanese SLE.

To determine how polymorphism of transporter associated with antigen processing 1 and 2 (TAP1 and 2) alleles contributed to the pathogenesis of systemic lupus erythematosus (SLE) in Japanese patients. TAP1 and TAP2 typing was carried out in 52 Japanese patients with SLE and 95 normal subjects by the PCR-RFLP (restriction fragment length polymorphism) method. HLA-DR typing and HLA-DRB1*15 genotyping were carried out by the PCR method and PCR-SSCP (single stranded DNA conformation polymorphism) method, respectively. No particular TAP 1 allele was associated with Japanese SLE or with immunological subgroup of SLE. TAP2H showed a tendency towards increased frequency in SLE (5.8% v 0% in control), but the corrected P value was not significant. No other particular association of TAP2 allele was observed. Furthermore, these was no evidence for linkage disequilibrium between any TAP1/TAP2 alleles and HLA-DRB1*1501--which is reported to be weakly but significantly association with Japanese SLE--in either the normal control or the SLE patient group. Neither the TAP1 nor the TAP2 gene appears to determine disease susceptibility to SLE in Japanese, and these results are in keeping with those reported in Caucasian SLE patients.

DNA single strand conformation polymorphism identifies five defined strains of hepatitis B virus (HBV) during an outbreak of HBV infection in an oncology unit.

An outbreak of hepatitis B virus (HBV) infection in a children's oncology unit was identified in which 61 children were shown to have been infected, 59 of them asymptomatically. In order to establish whether intra-unit cross infection had occurred, we used the single strand conformational polymorphism (SSCP) technique to analyse viral isolates from 57 of the infected children and 40 unrelated controls. HBV-specific primers were designed to amplify a 189 bp fragment of DNA encompassing part of the hypervariable pre-S1 region of the HBV genome. Denatured PCR products were compared after electrophoresis through polyacrylamide gels and staining with silver. By SSCP analysis, the unrelated infections each yielded a unique electrophoretic banding pattern, indicative of a variety of distinct virus strains. In contrast, most of the oncology patients had been infected with one of only five different strains. Three major groups comprising 19, 16, and 9 patients, respectively, and two minor groups of 5 and 3 patients were identified. Results indicate the occurrence of multiple episodes of cross infection, and demonstrate the sensitivity and value of SSCP as a technique to establish common sources of infection.

DNA single strand conformation polymorphism identifies five defined strains of hepatitis B virus (HBV) during an outbreak of HBV infection in an oncology unit

DNA single strand conformation polymorphism identifies five defined strains of hepatitis B virus (HBV) during an outbreak of HBV infection in an oncology unit

Single-stranded DNA conformation polymorphism at the Rdl locus in Hypothenemus hampei (Coleoptera: Scolytidae).

The homologue of the resistance to dieldrin gene (Rdl) in Drosophila melanogaster was cloned and sequenced in the scolytid beetle Hypothenemus hampei, a coffee pest resistant to cyclodiene insecticides in New Caledonia. The amino acid sequence of the Rdl exon no. 7 protein product in H. hampei was identical to that in D. melanogaster and showed the same amino acid change as that characterizing susceptible vs. resistant D. melanogaster. Samples from natural H. hampei populations (from Asia, the Pacific Islands, Africa and Central America), from reference susceptible (S) and resistant (R) laboratory strains, and from their hybrid progenies, were analysed at the Rdl locus using single-stranded DNA conformation polymorphism on polymerase chain reaction products. The susceptible allele was the only allele present in all samples from natural populations except in the only resistant population known to date (Ponerihouen, New Caledonia). Females and some males obtained at F1 from R x S crosses were heterozygous at the Rdl locus, confirming that this local mate competing species is diplo-diploid.

Analysis of single-strand DNA conformation polymorphism by capillary electrophoresis

Analysis of single-strand conformation polymorphism (SSCP) by capillary electrophoresis (CE) was developed. The conformational change of single-strand DNA is caused by a mutation in a DNA fragment. The change is detected as mobility shift in CE. The effects of acrylamide gel concentration, running temperature and fragment size amplified by the polymerase chain reaction (PCR) were studied to develop the separation of SSCP. The model DNA used was the divE 42 gene carrying wild- and mutant-type (G→A point mutation at the 141 site). The results show that two single-strand DNA fragments that differ in one nucleotide can be separated by CE within minutes. This method was also applied to the separation of SSCP for N- ras gene including four kinds of mutations. All mutations tested in this study could be distinguished. CE is well suited for clinical analysis of SSCP because it is rapid and reproducible, allows on-line detection and is easy.

ErbB-2 expression is correlated with poor prognosis for patients with osteosarcoma.

It has been reported that the c-erbB-2 protooncogene is frequently amplified and overexpressed in many types of cancers, except sarcomas and hematological malignancies. Expression of ErbB-2 in the tumors of 26 patients with conventional osteosarcoma was evaluated by immunoblotting. DNA from osteosarcoma tissues that expressed ErbB-2 were analyzed by Southern blot hybridization to examine gross rearrangement of the gene. The DNA was also surveyed for the presence of genetic mutation in the transmembrane domain of ErbB-2 by polymerase chain reaction-single-stranded DNA conformation polymorphism analysis. In addition, possible correlation of ErbB-2 expression with gender, age, histopathologic subtype, and response to chemotherapy was analyzed. Survival analysis was performed by the Kaplan-Meier test using the approximate chi-square statistic for the log-rank test. The ErbB-2 protein was detected in 11 of 26 osteosarcoma tissues (42%) by immunoblot analysis. Expression of ErbB-2 was confirmed by immunohistochemical studies using specific anti-ErbB-2 monoclonal antibody. However, neither amplification of the c-erbB-2 gene nor evidence of significant genetic mutation was found in these osteosarcomas. Expression of ErbB-2 examined by immunoblotting was most strongly correlated with early pulmonary metastases (P < 0.05). Among the entire group of 26 patients in this study, Kaplan-Meier life table survival of the patients with apparent ErbB-2 expression was significantly worse than that of the patients with little ErbB-2 expression (P < 0.01). In 42% of the osteosarcomas, the tumor cells expressed ErbB-2. Expression of ErbB-2 was strongly correlated with early pulmonary metastasis and poor survival rate for the patient. These data suggest that ErbB-2 plays a significant role in aggressive tumor growth and in the promotion of metastatic potential in osteosarcomas. ErbB-2 in the osteosarcoma tissues would be a useful prognostic marker for patients.

Detection of two different nonsense mutations in exon 44 of the PKD1 gene in two unrelated Italian families with severe autosomal dominant polycystic kidney disease.

Sixty-seven Italian patients with autosomal dominant polycystic kidney disease (ADPKD) were screened for mutations in the PKD1 gene. We used PCR, heteroduplex and single-strand conformation polymorphism DNA analysis, and automated DNA sequencing for exons 35, 36, 38, 44 and 45. We detected abnormal heteroduplexes in affected individuals from two unrelated families with clinically severe ADPKD phenotype. These changes were absent in other, unaffected members, as well as in the probands of the other families studied. DNA sequencing revealed in both cases different C to T transitions in exon 44, which created premature stop codons. Both mutations altered restriction sites, and the abnormal patterns were observed in all the affected family members. RT-PCR performed on lymphocyte mRNA showed that both the mutant and the normal transcript are represented. To our knowledge these are the first nonsense mutations described in the PKD1 gene.

The biochemical and phenotypic characterization of females homozygous for 5 alpha-reductase-2 deficiency.

The biochemical and physiologic manifestations of decreased 5 alpha-dihydrotestosterone (DHT) in females are characterized. Three females from the large Dominican kindred with 5 alpha-reductase-2 deficiency were identified as homozygous for a point mutation (R246W, C-->T) on exon 5 of the 5 alpha-reductase-2 gene by single strand DNA conformational polymorphism analysis and DNA sequence analysis. Body hair was decreased; there was no history of acne. Despite delayed menarche, all were fertile, and two had twins. Urinary 5 beta/5 alpha C19 and C21 steroid metabolite ratios were elevated. Plasma testosterone was normal to elevated, with low DHT, resulting in an increased testosterone/DHT ratio. 3 alpha,5 alpha-Androstanediol glucuronide was low. Menstrual cycle profiling performed in two subjects showed ovulatory gonadotropin peaks. Sebum production was normal. 5 alpha-Reductase-2-deficient homozygotic females demonstrate the importance of DHT in the physiology and pathophysiology of body hair growth. Normal sebum implies regulation by the 5 alpha-reductase-1 isoenzyme. Delayed puberty suggests involvement of 5 alpha-reductase-2 in menarche at the hypothalamic/pituitary and/or ovarian level. As two had nonidentical twins, DHT and/or the DHT/estradiol ratio may regulate follicular development, with lower levels permitting more than one dominant follicle per cycle and higher levels impairing follicular development and ovulation. Thus, females with 5 alpha-reductase-2 deficiency highlight a role for DHT in hirsutism and/or menstrual disorders.

Identification of Mutations by RNA Conformational Polymorphism “Bar Code” Analysis

DNA single-strand conformational polymorphism (SSCP) analysis is widely used for detection of point mutations in clinical specimens. Performing SSCP analysis with cRNA instead of DNA has been shown to improve mutation detection frequency. RNA can exist in numerous meta-stable conformations, which appear as patterns of bands on nondenaturing electrophoresis gels. Single base mutations can cause not only mobility shifts of major bands, but also loss of some conformations and appearance of new conformations. Unique RNA SSCP patterns associated with specific base sequences in many cases allow visual identification of point mutations. However, in some cases, the RNA SSCP pattern of a single base change in a sequence is not sufficiently different for a positive identification of the mutation. Improvement in the detection capability of RNA SSCP was obtained by adding 3′-deoxynucleotides to the transcription reaction. The presence of chain-terminating nucleotides in the transcription reaction formed numerous new RNA fragments, thereby generating complex band patterns (“bar codes”) unique to each RNA sequence. This method was applied to analyzing p53 mutations in patients with colon cancer.

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.

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.

Single-strand conformation polymorphisms can be used to detect T cell receptor gene rearrangements: an application to the in vivo hprt mutation assay

Epidemiologic application of the human in vivo hypoxanthine-guanine phosphoribosyltransferase (hprt) mutation assay requires screening of mutant colonies to differentiate independent from clonal origin. Previously, sibship was defined by Southern blot analysis of T cell receptor gene rearrangements. We report here a more expedient method to determine these rearrangements utilizing the polymerase chain reaction (PCR) and a DNA single-strand conformation polymorphism technique. The results are consistent with those obtained by Southern blotting in that sibship can be defined easily. A major advantage is that cells may be taken directly from the microtiter plate, eliminating the necessity to expand the clones and isolate genomic DNA. Cell lines which have not undergone receptor gene rearrangements cannot serve as PCR templates and do not interfere with this analysis. Furthermore, background from the large number of nonmutant lymphocytes present in the well does not hinder the analysis of the T cell receptor pattern of a mutant. This technique facilitates rapid screening of a large number of clones in a shorter time than Southern blotting, and is useful for the study of in vivo mutation and the clonal expansion of mutants in populations of T cells.