Detection of TP53 gene mutation in human meningiomas: A study using immunohistochemistry, polymerase chain reaction/single-strand conformation polymorphism and DNA sequencing techniques on paraffin-embedded samples ...

Minisatellite isoallelism, i.e. the occurrence of minisatellite alleles with different internal sequence composition but indistinguishable length, is a common limitation of minisatellite allele length analysis. Internal sequence variation can be used to distinguish such isoalleles, provided that detailed sequence knowledge of its basis is available. We now show that minisatellite isoalleles can also be simply resolved by single-stranded conformational polymorphisms (SSCP) arising during agarose gel electrophoresis. SSCP on agarose gels can be used to distinguish minisatellite isoalleles either after PCR amplification, or by standard Southern blot analysis of genomic DNA ...

OBJECTIVE: In this study, we examined the mutational spectrum of K-ras in cases of gallbladder and gallbladder carcinoma with an anomalous junction of the pancreaticobiliary duct (AJPBD). METHODS: We examined 35 gallbladders with AJPBD (20 with hyperplasia, 15 with carcinoma) and 38 gallbladders without AJPBD (lour normal gallbladders, four with hyperplasia, six with adenoma, 24 with carcinoma). Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and direct sequencing were performed to detect mutations in codon 12 or 13 of K-ras. RESULTS: In the eases with AJPBD, the prevalences of K-ras imitation were 15% (3/20) in hyperplasia, 60% (6/10) in stage I carcinoma, and 100% (5/5) in stage II-IV carcinoma. In the eases without AJPBD, the prevalences of K-ras mutation were 0% (0/4) in normal gallbladder, 0% (0/4) in hyperplasia, 17% (1/6) in adenoma, 7% (1/16) in stage I carcinoma, and 38% (3/8) in stage II-IV carcinoma. Prevalences of K-ras mutation in hyperplasia and ...

Longevity in livestock is a valuable trait. When productive animals live longer, fewer replacement animals need to be raised. However, selection for longevity is not commonly the focus of breeding programs as direct selection for long-lived breeding stock is virtually impossible until late in the reproductive life of the animal. Additionally the underlying genetic factors or genes associated with longevity are either not known, or not well understood. In humans, there is evidence that IGF 1 receptor (IGF1R) is involved in longevity. Polymorphism in the IGF1R gene has been associated with longevity in a number of species. Recently, 3 alleles of ovine IGF1R were identified, but no analysis of the effect of IGF1R variation on sheep longevity has been reported. In this study, associations between ovine IGF1R variation, longevity and fertility were investigated. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) was used to type IGF1R variation in 1,716 New Zeal ...

TY - JOUR. T1 - A case of simultaneous multiple gastric cancers with p53 gene mutation.. AU - Lin, S. Y.. AU - Lee, M. D.. AU - Wang, C. K.. AU - Chen, Y. J.. AU - Chang, J. G.. PY - 1994/1. Y1 - 1994/1. N2 - Simultaneous multiple gastric cancers are rarely observed in clinical practice, and its association with p53 gene mutation has not been mentioned in any previous reports. We report a case of advanced gastric cancer with two primary lesions in the stomach who received total gastrectomy. Tumor and surrounding normal tissues from the surgical specimen were studied by using polymerase chain reaction-single strand conformation polymorphism analysis, restriction enzyme digestion method and direct sequencing. Point mutations of p53 gene at codon 248 (CGG--,TGG) were found in both primary tumor foci. The patient developed cancerous peritonitis eight months after the operation and expired six months later. This report suggests that p53 gene mutation can occur at an earlier stage in the tumorigenesis ...

Background: DNA polymerases (Pols) represent potential candidates for cancer genes because of their central functions in DNA metabolism. Defects of some DNA Pols have shown cancer associations, but data on DNA polymerase (Pol) ε is limited. Materials and Methods: Twenty-four human breast cancer DNA samples and four control DNA samples were examined for possible mutation in the entire coding region of the 55 kDa small subunit of the human DNA Pol ε gene using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of the DNA and sequence analysis. In addition, 20 control DNAs were studied with PCR-SSCP for the end of intron 18 and exon 19 region. Results: An AATT deletion was found at one location in intron 18 in 2 out of the 24 breast cancer cases (8%), but in none of the control cases. In addition, a single base transition was found in the cancer DNAs in intron 14, but the same changes were also found in the control DNAs, suggesting polymorphism. Conclusion: ...

BACKGROUND Inactivation of the p53 tumor suppressor gene (also known as TP53) through a point mutation and/or loss of heterozygosity is one of the most common genetic changes found in various types of human tumors. PURPOSE Our purpose was to investigate the relationship between the presence of p53 gene mutations and survival of patients with non-small-cell lung cancer (NSCLC) of all stages who underwent surgery with preoperative curative intent as a routine therapeutic intervention. The prognostic significance of factors like sex, age, tumor histology, and the stage of the disease was also evaluated. METHODS We analyzed 120 tumor specimens from patients with histologically confirmed NSCLC for p53 mutations occurring in exons 5 through 8 by polymerase chain reaction-single-strand conformation polymorphism assay of genomic DNA. Univariate and multivariate analyses were performed to assess the association between p53 mutations and the survival of the NSCLC patients. RESULTS Fifty-one (43%) of 120

TY - JOUR. T1 - Cell cycle regulators in multiple myeloma. T2 - Prognostic implications of p53 nuclear accumulation. AU - Pruneri, Giancarlo. AU - Carboni, Nadia. AU - Baldini, Luca. AU - Intini, Daniela. AU - Colombi, Mariangela. AU - Bertolini, Francesco. AU - Valentini, Stefano. AU - Maisonneuve, Patrick. AU - Viale, Giuseppe. AU - Neri, Antonino. PY - 2003/1/1. Y1 - 2003/1/1. N2 - Multiple myeloma (MM) is characterized by a multistep process of tumorigenesis involving genes that control cell cycle progression. The prevalence and clinical implications of p53, p21, HDM-2, p27, and cyclin E immunoreactivity in MM patients, however, have not been fully elucidated. We evaluated the immunoreactivity (IR) for p53, p21, HDM-2, p27, cyclin E, and Ki-67 in bone marrow biopsies from 48 patients. In 34 (70.8%) cases, TP53 gene mutations and HDM-2 gene amplification were analyzed by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and Southern blot densitometric analyses in ...

Alterations in the FHIT gene region have been previously associated with smoking status and the occurrence of lung tumors. In the current study, we examined the nature of the mutations that occur at FHIT and the types of carcinogen exposures that are associated with FHIT alterations. We screened 40 primary lung tumors for the presence of point mutations within the coding exons of FHIT using PCr-single-strand conformational polymorphism. Tumors were also analyzed for allelic loss using microsatellite markers located in or near FHIT. No tumors contained point mutations within the coding region of the FHIT gene. However, several samples failed to generate a PCR product, suggesting that regions of the gene are homozygously deleted. Samples were reanalyzed for exon loss using PCR; 13 of 30 tumors failed to generate a PCR product, and 20 of 30 tumors were missing at least one FHIT exon or had loss (loss of heterozygosity or deletion) of one microsatellite marker, suggesting that regions of the gene ...

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TY - JOUR. T1 - Endoglin gene polymorphism as a risk factor for sporadic intracerebral hemorrhage. AU - Alberts, M. J.. AU - Davis, J. P.. AU - Graffagnino, C.. AU - McClenny, C.. AU - Delong, D.. AU - Granger, C.. AU - Herbstreith, M. H.. AU - Boteva, K.. AU - Marchuk, D. A.. AU - Roses, A. D.. PY - 1997/5. Y1 - 1997/5. N2 - Intracerebral hemorrhage (ICH) is a common and serious type of stroke. Recent studies have shown that inherited factors that affect the development of the vessel wall can increase the risk of ICH. We studied endoglin as a candidate gene in patients with sporadic ICH, since mutations in this gene can cause telangiectasia formation. One hundred three patients with sporadic ICH and 202 controls were studied. The polymerase chain reaction and single- strand conformational polymorphism analysis were used to screen for mutations in exon 7 of the endoglin gene. No coding mutations in exon 7 were identified in the ICH patients or controls. A 6-base intronic insertion was found 26 ...

Alterations of the p53 gene and the p53 protein are common in a wide spectrum of human malignancies. In several tumor types, p53 gene mutation and/or p53 protein overexpression correlate with a more clinically aggressive phenotype as judged by worse patient survival. This has not been clearly demonstrated to be the case in colorectal cancer. Herein, we report results of the prognostic significance of p53 protein accumulation and gene mutation in a large series of colorectal cancers (n = 541) with long patient follow-up (mean, 87 months). The large majority of patients (95%) received no postoperative systemic adjuvant therapy. The incidence of p53 accumulation detected by immunohistochemistry with the monoclonal antibody DO-7 was 30%, whereas the incidence of p53 gene mutation in exons 5-8 detected using PCR-single strand conformation polymorphism was 36%. Accumulation of p53 protein was associated with improved patient survival independent of tumor stage or grade (hazard ratio, 0.66; 95% ...

The aetiology and detection of human herpes virus type 8 (HHV-8) DNA sequences in Kaposis sarcoma (KS) is a matter of intense investigation. We report on the detection of HHV-8 DNA and sequence polymorphism in different clinicopathological subtypes of cutaneous KS samples from South Africa. The diagnosis was confirmed by histological examination in all cases. Six patients had classic KS (CKS), 3 epidemic KS (EKS), and 3 iatrogenic KS (IKS). A nested polymerase chain reaction (PCR) assay was used to detect HHV-8 DNA in cell lysates, prepared from formalin fixed, paraffin embedded sections. We investigated polymorphism in the HHV-8 DNA using single-stranded conformational polymorphism (SSCP) analysis on the PCR products, followed by direct sequencing. HHV-8 DNA was detected in all the patients with KS, irrespective of the clinicopathological subtype. Direct sequencing was performed on 5 selected cases and showed single base pair substitutions in all. The spectrum of mutations was similar to those ...

The Mycobacterium tuberculosis H37Rv efpA gene encodes a putative efflux protein, EfpA, of 55,670 Da. The deduced EfpA protein was similar in secondary structure to Pur8, MmrA, TcmA, LfrA, EmrB, and other members of the QacA transporter family (QacA TF) which mediate antibiotic and chemical resistance in bacteria and yeast. The predicted EfpA sequence possessed all transporter motifs characteristic of the QacA TF, including those associated with proton-antiport function and the motif considered to be specific to exporters. The 1,590-bp efpA open reading frame was G+C rich (65%), whereas the 40-bp region immediately upstream had an A+T bias (35% G+C). Reverse transcriptase-PCR assays indicated that efpA was expressed in vitro and in situ. Putative promoter sequences were partially overlapped by the A+T-rich region and by a region capable of forming alternative secondary structures indicative of transcriptional regulation in analogous systems. PCR single-stranded conformational polymorphism ...

Hybridization analysis showed a p27 gene region, which exhibits different patterns with two probes derived from two biological distinct CTV isolates. In an attempt to screen whether that gene region differs in mild and severe strains, six CTV isolates belonging to different biogroups were compared for variations in their p27 gene by analysis of single-strand conformation polymorphism (SSCP). The p27 gene was reverse transcribed and amplified by PCR and thirty clones of each isolate were obtained. From each clone, two fragments of the gene were amplified by PCR: fragment (a), 459 bp long, and fragment (b), 281 bp long. Sequence variations in both gene fragments were studied by SSCP analysis. A variety of SSCP patterns was obtained from each isolate, being isolates belonging to the groups II-IV and III those with the higher and lower number of them. Moreover, SSCP analysis provided a rapid procedure to screen the genetic heterogeneity of the viral isolate reducing considerably the amount of ...

Aim: To determine the frequency and nature of mutations in the gene ABCA4 in a cohort of patients with bulls-eye maculopathy (BEM).. Methods: A panel of 49 subjects (comprising 40 probands/families, 7 sibling pairs and a set of three sibs) with BEM, not attributable to toxic causes, was ascertained. Blood samples from each patient were used to extract genomic DNA, with subsequent mutation screening of the entire coding sequence of ABCA4, using single-strand conformational polymorphism (SSCP) analysis and direct sequencing.. Results: Fourteen probands (35%) were found to have a potentially disease-causing ABCA4 sequence variant on at least one allele. Three patients had a Gly1961Glu missense mutation, the most common variant in Stargardt disease (STGD), with 2 of these subjects having a macular dystrophy (MD) phenotype and a second ABCA4 variant previously associated with STGD. The second most common STGD mutation, Ala1038Val, was seen in one patient with cone-rod dystrophy (CORD). Five novel ...

TY - JOUR. T1 - The dithiacyclooctane cation (DTCO+). T2 - Conformational analysis, interconversion barriers and bonding. AU - Stowasser, Ralf. AU - Glass, Richard S. AU - Hoffmann, Roald. PY - 1999/7. Y1 - 1999/7. N2 - A theoretical conformation analysis of the dithiacyclooctane radical cation (DTCO+) suggests that the lowest energy conformer is a chair-boat, with a partial but significant S-S σ bond. For the ring flip process of this molecule we calculate a barrier of 40 kJ mol-1 and two possible pathways: one involves a boat-boat conformer and an untwisted transition structure, the other a chair-chair conformer and a twisted transition structure.. AB - A theoretical conformation analysis of the dithiacyclooctane radical cation (DTCO+) suggests that the lowest energy conformer is a chair-boat, with a partial but significant S-S σ bond. For the ring flip process of this molecule we calculate a barrier of 40 kJ mol-1 and two possible pathways: one involves a boat-boat conformer and an ...

The present study was aimed at evaluating random bred broiler and layer lines maintained at Directorate of Poultry Research, Rajendranagar, Hyderabad with respect to expression profiling, epigenetic and functionality of promoter of BMP3 and BMP4 genes. BMP3 acts as a negative regulator for osteogenesis and as a modulator of osteogenic activity of other BMPs in vivo while BMP4 shows osteogenetic activity in vivo and in vitro. The functionality of promoters was elucidated by means of reporter GFP marker in vivo. BMP3 gene promoter was divided into two fragments of 501 and 786 bp while that of BMP4 was divided into fragments of 541 bp and 1.1 kb size. Larger fragments were better promoters. Both the promoters displayed polymorphism by means of single strand conformation polymorphism study using blood samples of broiler (286) and layer (270) birds. For BMP3 gene promoter, haplotype, h1 was predominant (0.735 and 0.62) and h3 was the least frequent (0.046 and 0.04) in broiler and layer lines, ...

We have previously found an inverse association of childhood asthma and fungal diversity by classical culturing (Ege et al. NEJM 2011;364:701-9), which covers only a small minority of taxa.. The aim of the present analysis was to include also non-cultivable fungi by a molecular technique. For this purpose we applied single strand conformation polymorphism polymerase chain reaction (SSCP-PCR) targeting the internal transcribed spacer region (ITS) of fungi (Janke et al. Curr Microbiol 2013;67:156-69).. We used data from the GABRIELA study and analysed mattress dust samples from 844 Bavarian children age 6-10 years. DNA was extracted from dust samples, and the fungus-specific ITS region was amplified by PCR. The amplicons were subjected to non-denaturing gel-electrophoresis, and the resulting band patterns were digitized and normalized. We applied a factor analysis for the identification of the relevant gel positions and explored associations with a lifetime physicians diagnosis of asthma by ...

We reported an association of smoking-induced lung cancer susceptibility with the human cytochrome P450 1A1 (CYP1A1) polymorphisms in our previous studies. To investigate a relationship between genetically determined individual predispositions and mutations of target genes in the early stage of lung carcinogenesis, we examined p53 mutations in relation to germ line polymorphisms of the CYP1A1 and GSTM1 genes, using surgical specimens of 148 non-small cell lung cancer patients who were smokers. The frequency of p53 mutations among heavy smokers was higher than in patients who had never smoked [P , 0.01; odds ratio (OR), 3.74; 95% confidence interval (CI), 1.46-9.56]. By single-strand conformational polymorphism, aberrant migration bands of p53 gene fragments were detected in 56 cases (38%). Smokers with susceptible rare homozygous alleles of either the MspI or Ile-Val polymorphism of the CYP1A1 gene have a 4.5-fold (P , 0.005; OR, 4.48; 95% CI, 1.64-12.26) or 5.5-fold (P , 0.01; OR, 5.52; 95% CI, ...

Features of our method make it inherently robust. First, double-end labeling allows for independent detection of the two cleavage products in different fluorescence channels, which guards against false positives. Second, we reamplify and retest each individual from a pool in the same way, which further eliminates false positives. Third, from fragment mobilities we estimate the position of the mutation within the fragment; we find that this estimation is accurate to ±10 bp (data not shown). We use both the near certainty of a mutation being in the fragment and the mobility information to help in identifying the heterozygous changes. Other technologies have been applied to single test samples and have demonstrated high accuracy in detection of heterozygotes (Spiegelmanet al. 2000; Kwok 2001; Liet al. 2002). However, methods that simply report the presence of a migration or retention anomaly, such as single-strand conformational polymorphism, temperature-gradient capillary electrophoresis, and ...

The respiratory mycobiome is an important but understudied component of the human microbiota. Like bacteria, fungi can cause severe lung diseases, but their infection rates are much lower. This study compared the bacterial and fungal communities of sputum samples from a large cohort of 56 adult patients with cystic fibrosis (CF) during nonexacerbation periods and under continuous antibiotic treatment. Molecular fingerprinting based on single-strand conformation polymorphism (SSCP) analysis revealed fundamental differences between bacterial and fungal communities. Both groups of microorganisms were taxonomically classified by identification of gene sequences (16S rRNA and internal transcript spacer), and prevalences of single taxa were determined for the entire cohort. Major bacterial pathogens were frequently observed, whereas fungi of known pathogenicity in CF were detected only in low numbers. Fungal species richness increased without reaching a constant level (saturation), whereas bacterial ...

Highlights Helical Molecules and Aggregates Molecules with Helical Structure: How To Build a Molecular Spiral Staircase Carsten Schmuck* Keywords: arenes · conformation analysis · foldamers · helical structures Helical Compounds Molecules with helical structure have fascinated chemists for may years now. Due to their nonplanar structure, such molecules are inherently chiral and exhibit interesting optical and electronic properties. In principle, molecules with such unusual topologies[1] can be synthesized through three different approaches: First, in rigid molecules steric effects can be used to enforce a helical structure. This is the case, for example, in the long-known and well-studied helicenes and their derivatives.[2] An increasing ortho annulation of aromatic ring systems first causes an increasing steric interaction between the H atoms on the terminal rings, leading to a nonplanar conformation. When the number of annulated arenes further increases, the terminal rings will overlap, ...

زمینه مطالعاتی: انتخاب به وسیله ژنتیک مولکولی روی ژن-های منحصر بفرد یک روش مطمئن برای بهبود ژنتیکی صفات مهم اقتصادی در حیوانات اهلی می-باشد. صفت چندقلوزایی یکی از صفات مهم اقتصادی در صنعت گوسفندداری می-باشد که در سال-های اخیر توجه متخصصین اصلاح نژاد را به خود جلب کرده است. ژن فاکتور رشد و تمایز شماره 9 (GDF9) از مهم-ترین ژن-های کاندیدای موثر بر صفت چندقلوزایی در گوسفند می-باشد. هدف: این آزمایش جهت بررسی وجود چند شکلی در جایگاه نیمه دوم (منتهی به3) اگزون 2 ژن GDF9 گوسفند نژاد کرمانی انجام شد. روش کار: در این مطالعه، از 102 رأس گوسفند خونگیری شد. پس از استخراج DNA، تعیین

Pyrazinamide is a first-line drug for treating tuberculosis, but pyrazinamide resistance testing is usually too slow to guide initial therapy, so some patients receive inappropriate therapy. We therefore aimed to optimize and evaluate a rapid molecular test for tuberculosis drug resistance to pyrazinamide. Tuberculosis polymerase chain reaction single strand conformational polymorphism (PCR-SSCP) was optimized to test for mutations causing pyrazinamide resistance directly from sputum samples and Mycobacterium tuberculosis isolates. The reliability of PCR-SSCP for sputum (n=65) and Mycobacterium tuberculosis isolates (n=185) from 147 patients was compared with four tests for pyrazinamide resistance: Bactec-460 automated-culture; the Wayne biochemical test; DNA sequencing for pncA mutations; and traditional microbiological broth culture. PCR-SSCP provided interpretable results for 96% (46/48) of microscopy-positive sputum samples, 76% (13/17) of microscopy-negative sputa and 100% of Mycobacterium ...

Acetyl-coenzyme A carboxylase α (ACC-alpha) is considered as the key regulatory enzyme in fatty acid biosynthesis. ACC-alpha gene is located on Caprine chromosome 11 and is polymorphic in many goat breeds. In the current study, we aimed to find possible single nucleotide polymorphisms (SNPs) in the exon 1 region of the ACC-alpha gene in Iranian Mahabadi goat breed. Genomic DNA was extracted from blood samples of 150 Mahabadi does. The exon 1 region of the ACC-alpha gene was amplified to produce a 390 bp fragment. The PCR products were analyzed by both polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) techniques. RFLP was performed utilizing HinfI endonuclease enzyme. No polymorphism was observed after digestion of the PCR products using HinfI. However, SSCP of the PCR products revealed two conformation patterns at the exon 1 region of goat ACC-alpha gene with frequencies of 86% and 14%,

Marfan syndrome is a dominantly inherited connective tissue disorder with a wide range of phenotypic severity. The condition is the result of mutations in FBN1, a large gene composed of 65 exons encoding the fibrillin-1 protein. While mutations causing classic manifestations of Marfan syndrome have been identified throughout the FBN1 gene, the six previously characterized mutations resulting in the severe, perinatal lethal form of Marfan syndrome have clustered in exons 24-32 of the gene. We screened 8 patients with either neonatal Marfan syndrome or severe cardiovascular complications of Marfan syndrome for mutations in this region of the gene. Using intron-based exon-specific primers, we amplified exons 23-32 from genomic DNAs, screened these fragments by single-stranded conformational polymorphism analysis, and sequenced indicated exons. This analysis documented mutations in exons 25-27 of the FBN1 mutations in 6 of these patients. These results, taken together with previously published FBN1 ...

TY - JOUR. T1 - Mutation analysis of neurofilament-light gene in Chinese Charcot-Marie-Tooth disease. AU - Luo, Wei. AU - Tang, Bei Sha. AU - Zhao, Guo Hua. AU - Li, Qi. AU - Xiao, Jianfeng. AU - Yang, Qi Dong. AU - Xia, Jia Hui. PY - 2003/4/1. Y1 - 2003/4/1. N2 - Objective: To study the characteristic of the mutation of neurofilament-light (NF-L) gene in Chinese Charcot-Marie-Tooth disease (CMT) patients. Methods: Mutation analysis of NF-L gene was made by use of polymerase chain reaction-single strand conformation polymorphsim combined with DNA direct sequencing in 32 CMT probands from the Hans of five provinces in China who had been diagnosed by clinical feature and electromyography and/or biopsy of sural nerve. Results: In 32 CMT probands, only one sporadic case was found to display variant banding pattern, and this case was confirmed as 1329C to T (Tyr443Tyr) single nucleotide polymorphism by sequencing. Conclusion: Mutation of NF-L gene may be rare in Chinese CMT patients.. AB - Objective: ...

TY - JOUR. T1 - Cultivation of mesophilic soil crenarchaeotes in enrichment cultures from plant roots. AU - Simon, Holly M.. AU - Jahn, Courtney E.. AU - Bergerud, Luke T.. AU - Sliwinski, Marek K.. AU - Weimer, Paul J.. AU - Willis, David K.. AU - Goodman, Robert M.. PY - 2005/8. Y1 - 2005/8. N2 - Because archaea are generally associated with extreme environments, detection of nonthermophilic members belonging to the archaeal division Crenarchaeota over the last decade was unexpected; they are surprisingly ubiquitous and abundant in nonextreme marine and terrestrial habitats. Metabolic characterization of these nonthermophilic crenarchaeotes has been impeded by their intractability toward isolation and growth in culture. From studies employing a combination of cultivation and molecular phylogenetic techniques (PCR-single-strand conformation polymorphism, sequence analysis of 16S rRNA genes, fluorescence in situ hybridization, and real-time PCR), we present evidence here that one of the two ...

The Ataxia-telangiectasia mutated (ATM) gene encodes a multifunctional kinase, which is linked to important cellular functions. Women heterozygous for ATM mutations have an estimated relative risk of developing breast cancer of 3.8. However, the pattern of ATM mutations and their role in breast cancer etiology has been controversial and remains unclear. In the present study, we investigated the frequency and spectrum of ATM mutations in a series of sporadic breast cancers and controls from the Brazilian population. Using PCR-Single Strand Conformation Polymorphism (SSCP) analysis and direct DNA sequencing, we screened a panel of 100 consecutive, unselected sporadic breast tumors and 100 matched controls for all 62 coding exons and flanking introns of the ATM gene. Several polymorphisms were detected in 12 of the 62 coding exons of the ATM gene. These polymorphisms were observed in both breast cancer patients and the control population. In addition, evidence of potential ATM mutations was observed in 7

BACKGROUND. Microsatellite instability (MI) is a frequent occurrence in endometrioid carcinoma of the endometrium (EC). Several genes known to contain mononucleotide short tracts in their coding sequences (TGF-β RII, IGFIIR, BAX, hMSH6, and hMSH3) are likely targets for mutations in these tumors. METHODS. DNA from 24 patients with EC and MI was extracted from blood and from fresh-frozen and paraffin embedded tumor tissue. Seven of these patients were found to have metastatic spread to paraaortic lymph nodes. DNA also was studied from 10 patients with EC without MI. RESULTS. Frameshift mutations at coding mononucleotide repeats were detected by single strand conformation polymorphism analysis and DNA sequencing. Frameshift mutations were detected more frequently in BAX (11 of 24 MI positive (+) tumors; 45.8%) than in TGF-β RII (0 of 24 tumors; 0%), IGFIIR (3 of 24 tumors; 12.5%), hMSH3 (6 of 24 tumors; 25%), or hMSH6 (0 of 24 tumors; 0%). The mutations frequently were distributed ...

Background: Folylpolyglutamate synthetase (FPGS), an important enzyme in the folate metabolic pathway,plays a central role in intracellular accumulation of folate and antifolate in several mammalian cell types. Loss ofFPGS activity results in decreased cellular levels of antifolates and consequently to polyglutamatable antifolatesin acute lymphoblastic leukemia (ALL). Materials and Methods: During May 1997 and December 2003, 134children diagnosed with ALL were recruited from one hospital in Thailand. We performed a mutation analysis inthe coding regions of the FPGS gene and the association between single nucleotide polymorphisms (SNPs) withinFPGS in a case-control sample of childhood ALL patients. Mutation screening was conducted by polymerasechain reaction-single strand conformation polymorphism (PCR-SSCP) and subsequently with direct sequencing(n=72). Association analysis between common FPGS variants and ALL risk was done in 98 childhood ALL casesand 95 healthy volunteers recruited as controls.

We have recently isolated a human gene, ROX, encoding a new member of the basic helix-loop-helix leucine zipper protein family. ROX is capable of heterodimerizing with Max and acts as a transcriptional repressor in an E-box-driven reporter gene system, while it was found to activate transcription in HeLa cells. ROX expression levels vary during the cell cycle, being down-regulated in proliferating cells. These biological properties of ROX suggest a possible involvement of this gene in cell proliferation and differentiation. The ROX gene maps to chromosome 17p13.3, a region frequently deleted in human malignancies. Here we report the genomic structure of the human ROX gene, which is composed of six exons and spans a genomic region of less than 40 kb. In an attempt to identify possible inactivating mutations in the ROX gene in human breast cancer, we performed a single-strand conformation polymorphism analysis of its coding region in 16 sporadic breast carcinomas showing loss of heterozygosity in ...

Background. Human and animal studies support the role of MC4R and MC3R in human obesity, but limited data are available on the genetic contribution to obesity in South African populations. Objective. To screen obese-overweight South African pupils for MC3R and MC4R polymorphisms that may play a role in the development of obesity. Design. A cross-sectional study screened 227 obese-overweight (115 black and 112 coloured) and 204 normal weight (94 black, 110 coloured) school pupils for the presence of MC4R and MC3R polymorphisms using a single strand conformation polymorphism, subsequent sequencing, and allele specific restriction enzyme analysis. Results. Two polymorphisms were detected in the MC3R (T6K and V81I) but none in MC4R. After adjusting for age, gender and case-control status, the frequency distributions of T6K and V81I genotype and allele varied significantly between the ethnic groups. The frequency of the V81I A allele was significantly lower in coloured overweight-obesity than normal pupils

Conclusions Imagine • Innovate • Integrate The NSET procedure is effective for uterine transfer of blastocyst stage embryos. Surgery causes a significant.Neutering companion exotic mammals (Proceedings). Once the uterine horn has been. A single interrupted suture.Alpaca Article. An Introduction to. The uterus has a short body with two horns,. Administered as a single 1ml IM injection it will allow return to normal.uterine body and uterine horn) were taken at slaughter for the analysis of gene ex-. (Single Strand Conformation Polymorphism), HRM (High Resolution Melting), se-.. ovarian duct definition, meaning, English dictionary, synonym, see also Ontarian,ovary,ovaritis,ova, Reverso dictionary, English definition, English vocabulary.Open pulled straw vitrification of murine and caprine embryos and timed deep uterine insemination of goats Dissertation to obtain the Ph. D. degree.proliferate rapidly and, as a result, large numbers of cells can be produced from a single embryo within. ...

Principal Investigator：MORISHITA Fujio,一瀬 光之尉, Project Period (FY)：1993 - 1994, Research Category：Grant-in-Aid for General Scientific Research (B), Research Field：工業分析化学

Epithelial (E)-cadherin and its associated cytoplasmic proteins (alpha-, beta-, and gamma-catenins) are important mediators of epithelial cell-cell adhesion and intracellular signaling. Much evidence exists suggesting a tumor/invasion suppressor role for E-cadherin, and loss of expression, as well as mutations, has been described in a number of epithelial cancers. To investigate whether E-cadherin gene (CDH1) mutations occur in colorectal cancer, we screened 49 human colon carcinoma cell lines from 43 patients by single-strand conformation polymorphism (SSCP) analysis and direct sequencing. In addition to silent changes, polymorphisms, and intronic variants in a number of the cell lines, we detected frameshift single-base deletions in repeat regions of exon 3 (codons 120 and 126) causing premature truncations at codon 216 in four replication-error-positive (RER+) cell lines (LS174T, HCT116, GP2d, and GP5d) derived from 3 patients. In LS174T such a mutation inevitably contributes to its lack of E

Although postoperative chemotherapy is widely accepted as the standard modality for Dukes stage C or earlier stage colorectal cancer (CRC) patients, biomarkers to predict those who may benefit from the therapy have not been identified. Previous in vitro and clinical investigations reported that CRC patients with wild-type p53 gene (TP53)-tumors benefit from 5-fluorouracil (5-FU) based chemotherapy, while those with mutated TP53-tumors do not. However, these studies evaluated the mutation-status of TP53 by immunohistochemistry with or without single-strand conformation polymorphism, and the mutation frequency was different from study to study. In addition, the polymorphic status at p53 codon 72, which results in arginine or proline residues (R72P) and is thought to influence the function of the protein significantly, was not examined. To evaluate the significance of the TP53 mutation as a molecular marker to predict the prognosis of CRC patients, especially those who received postoperative chemotherapy,

The growth hormone gene could be an attractive candidate gene for milk production in goats. Single-strand conformation polymorphism was used to identify polymorphism at the goat growth hormone (gGH) gene. For this purpose, genotyping of 90 Talli goat breeds was performed. Nine conformational patterns were observed in exon 4 of the gGH gene, with frequencies of 27.7% for the homozygous pattern (AA) and 72.2% for all of other heterozygous patterns (A\B, A\C, A\B\C, A\B\D\E, A\B\C\F, A\C\F, A\B\E, A\B\F). The results showed that exon 4 of the GH gene in Talli goats is highly polymorphic ...

What you need to do is cut out the individual SSCP bands out of hte acrylamide gel and rePCR them and then sequence those PCR products. Mark the original gel with some radioactive ink and reexpose it on film. Using the marks, align the film and the gel to precisely cut out the gel bands. Soak the cut out bands in 50 ul 10 mM Tris and use 5 ul in a new PCR reaction. When you sequence these reactions, you will only be seeing one allele instead of two, so heterozygosity wont hide the mutation. Also, you should try MDE gel solution + 5% glycerol for SSCP gels instead of acrylamide - your mutations will show up a lot easier. beth : , I am new to this newsgroup, please forgive me if my behavir is wrong : , or : , rude. : , I am looking for a mutation site of BRCA1 and BRCA2 gene, the : , material : , of DNA is mostly coming from paraffin. : , I have done SSCP for hundreds of times ,some of them indicate the : , mutation definitaly. However, the results of DNA sequence always beat : , me:there is no ...

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COL4A3/COL4A4 mutations: From familial hematuria to autosomal-dominant or recessive Alport syndrome. BACKGROUND: Mutations of the type IV collagen COL4A5 gene cause X-linked Alport syndrome (ATS). Mutations of COL4A3 and COL4A4 have been reported both in autosomal-recessive and autosomal-dominant ATS, as well as in benign familial hematuria (BFH). In the latter conditions, however, clinical features are less defined, few mutations have been reported, and other genes and non-genetic factors may be involved. METHODS: We analyzed 36 ATS patients for COL4A3 and COL4A4 mutations by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and direct sequencing. Sporadic patients who had tested negative for COL4A5 mutations were included with typical cases of autosomal recessive ATS to secure a better definition of the phenotype spectrum. RESULTS: We identified seven previously undescribed COL4A3 mutations: in two genetic compounds and three heterozygotes, and one in COL4A4. In ...

BACKGROUND AND PURPOSE: Plasma glutathione peroxidase (GPx-3)-deficiency increases extracellular oxidant stress, decreases bioavailable nitric oxide, and promotes platelet activation. The aim of this study is to identify polymorphisms in the GPx-3 gene, examine their relationship to arterial ischemic stroke (AIS) in a large series of children and young adults, and determine their functional molecular consequences. METHODS: We studied the GPx-3 gene promoter from 123 young adults with idiopathic AIS and 123 age- and gender-matched controls by single-stranded conformational polymorphism and sequencing analysis. A second, independent population with childhood stroke was used for a replication study. We identified 8 novel, strongly linked polymorphisms in the GPx-3 gene promoter that formed 2 main haplotypes (H1 and H2). The transcriptional activity of the 2 most prevalent haplotypes was studied with luciferase reporter gene constructs. RESULTS: The H2 haplotype was over-represented in both patient ...

Background: The X-linked form of chronic granulomatous disease (CGD) is a primary immunodeficiency that affects phagocytes of the innate immune system and is characterized by an increased susceptibility to severe bacterial and fungal infections. It is caused by mutations in the CYBB gene, which encodes the 91-kD subunit of phagocyte NADPH oxidase. Aim: To identify the mutation in the CYBB gene in two unrelated patients from Chile with, the diagnosis of X-linked CGD and their families. Patients and methods: The molecular genetic defects of two unrelated patients from Chile with X-linked CGD caused by defects in the CYBB gene were investigated. The underlying mutation was investigated by single strand conformation polymorphism (SSCP) analysis of PCR-amplified genomic DNA and by sequencing of the affected gene region. Results: We found an insertion c.1267_1268insA in exon 10 leading to a frameshift mutation. This mutation is a novel report. We also identified a splice site mutation in the other ...

Abstract: : Purpose: Define the role of MYOC, CYP1B1 and PITX2 in patients with juvenile open angle glaucoma. Methods: We undertook mutational analysis of three glaucoma-related genes (MYOC, CYP1B1 and PITX2)using a combination of single strand conformation polymorphism (SSCP), and direct cycle sequencing. The patient population included 60 unrelated cases affected with JOAG or early onset glaucoma (onset age 5-40 years). Results: MYOC mutations were identified in 8/60 individuals (13.3%); CYP1B1 mutations in 3/60 (5%) and none in PITX2. Individuals with MYOC mutations showed greater phenotypic variability than expected, and included pigment dispersion syndrome and a mixed-mechanism glaucoma. Mutations in CYP1B1 were identified in three cases that did not have features suggestive of congenital glaucoma. Study of one large pedigree with autosomal dominant glaucoma demonstrated segregation of both MYOC and CYP1B1 mutations with the disease. Those who carried the MYOC mutation alone had an average ...

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ), a strong mutagen/carcinogen, belongs to a group of heterocyclic amines that are formed (ng/g amounts) during the cooking of protein containing food. The mutational specificity of IQ in Escherichia coli was determined in a forward mutation assay using the yeast URA3 gene as a target. The plasmid pTU-AC, containing the target URA3, was randomly modified in vitro using N-hydroxy-IQ, and subsequently transformed into an E. coli pyrF strain (DB6656). Mutant clones were directly selected by their ability to grow on medium containing 5-fluoro-orotic acid which is toxic to URA3+ clones and thereby selects for URA3- mutants. Single Strand Conformation Polymorphism (SSCP) was used to map the mutation-containing regions of URA3, so that it was necessary to sequence only the relevant, mutation-containing fragment and not the entire gene. At a modification level of 7 IQ-lesions/URA3 gene, the predominant mutations were base substitutions (approximately 70%), ...

Background: The PARK2 gene at 6q26 encodes parkin, whose inactivation is implicated in an early-onset autosomal recessive form of Parkinson disease (PD). Objective: To evaluate the influence of heterozygosity for parkin mutation on onset age in a sample of families with at least 2 PD-affected members. Design: Clinical and genetic study. Setting: Twenty collaborative clinical sites. Patients: Patients with familial PD collected in the GenePD study. Studied families were selected for (1) affected sibling pairs sharing 2 alleles identical by state at PARK2 (D6S305) or (2) 1 or more family members with onset age younger than 54 years, regardless of D6S305 status. At least 1 member from each of 183 families underwent comprehensive screening for deletion/insertion variants and point mutations in PARK2. Main Outcome Measures: Mutations in the parkin gene were screened by means of single-stranded conformation polymorphism and sequencing in all 12 coding exons and flanking intronic sequences for point ...

The glp-1 gene product mediates cell-cell interactions required for cell fate specification during development in Caenorhabditis elegans. To identify genes that interact with glp-1, we screened for dominant suppressors of two temperature-sensitive glp-1 alleles and recovered 18 mutations that suppress both germline and embryonic glp-1 phenotypes. These dominant suppressors are tightly linked to glp-1 and do not bypass the requirement for a distal tip cell, which is thought to be the source of a signal that is received and transduced by the GLP-1 protein. Using single-strand conformation polymorphism (SSCP) analysis and DNA sequencing, we found that at least 17 suppressors are second-site intragenic revertants. The suppressors, like the original glp-1(ts) mutations, are all located in the cdc10/SWI6/ankyrin domain of GLP-1. cdc10/SWI6/ankyrin motifs have been shown to mediate specific protein-protein interactions in other polypeptides. We propose that the glp-1(ts) mutations disrupt contact between GLP-1

Mutations in the p53 tumor suppressor gene and K-ras oncogene have been frequently found in sputum and bronchoalveolar lavage (BAL) samples of lung cancer patients and also in those of patients prior to presenting clinical symptoms of lung cancer, suggesting they may provide useful biomarkers for early lung cancer diagnosis. However, the detection of these gene mutations in sputum and BAL samples has been complicated by the fact that they often occur in only a small fraction of epithelial cells among sputum cells and, in the case of p53 gene, at many codons. In this study, sputum cells were collected on a filter membrane by sputum cyto-centrifugation and morphologically analyzed. Epithelial cells were selectively taken by using a laser capture microdissection microscope and analyzed by polymerase chain reaction (PCR) and single-stranded conformational polymorphism (SSCP), for p53 mutations, and PCR and denaturing gradient gel electrophoresis (DGGE), for K-ras mutations. This method was applied ...

AIMS: To attempt to detect p53 gene mutations in the plasma of patients with large bowel carcinoma. METHODS: Plasma was collected from 20 control patients with no history of cancer and from 17 patients with large bowel carcinoma. Corresponding tumour and benign lymph node (control) samples for each of the carcinoma patients were obtained from paraffin blocks. A Dukes stage was determined for each tumour. DNA was extracted from the plasma samples and the paraffin embedded tissue using previously described methods. A nested primer polymerase chain reaction protocol was used for the amplification of exons 5 to 8 of the p53 gene. Cold single strand conformational polymorphism (SSCP) was performed on mini gels and silver stained. Abnormal bands were excised, the DNA eluted, and reamplified for automated dye termination sequencing. Any sample showing an apparent mutation was rechecked from the original extracted DNA sample at least three times. RESULTS: p53 gene mutations were not found in the ...

Includes bibliographical references and index. Nucleic acid methods: The manipulation of nucleic acids : basic tools and techniques / Gayle Corkill and Ralph Rapley. Restriction enzymes : tools in clinical research / Gareth J.S. Jenkins. Principles and medical applications of the polymerase chain reaction / Bimal D.M. Theophilus. Probe design, production, and applications / Marilena Aquino de Muro. Southern blotting as a diagnostic method / Bronwen M. Harvey and Pirkko Soundy. Capillary electrophoresis of DNA : biomedical applications / Beatriz Sanchez-Vega. Denaturing high-performance liquid chromatography (DHPLC) for nucleic acid analysis / Kim Hung Leung and Shea Ping Yip. Denaturing gradient gel electrophoresis (DGGE) / Jeroen H. Roelfsema and Dorien J.M. Peters. Single strand conformation polymorphism (SSCP) analysis / Kim Hung Leung and Shea Ping Yip. Randomly amplified polymorphic DNA (RAPD) : a useful tool for genomic characterization of different organisms / Lúcia Maria da Cunha ...

To confirm the diagnosis of CAA at the molecular level, a mutational analysis of the ALB was carried out as described in Materials and methods. In this case the combination of heteroduplex analysis and single-strand conformation polymorphism did not allow an unambiguous localisation of the mutation site. Therefore all the fourteen coding exons and their adjacent intron regions were submitted to DNA sequence analysis, which clearly indicated the presence of a single mutation in the region encompassing exon 12 and the intron 11 - exon 12 and exon 12 - intron 12 junctions, that escaped our electrophoretic analysis. The electropherogram of the proband revealed that she is homozygous for a G,A transition at position c.1652+1, the first base of intron 12 in a 5 GT consensus sequence (Figure 3a). Both parents were heterozygous for the wild-type and mutated alleles, as the sequencing electropherograms showed the presence of two superimposed peaks at nucleotide c.1652+1 (Figure 3b and 3c). To establish ...

Long QT syndrome (LQT) is an inherited cardiac disorder that causes syncope, seizures and sudden death from ventricular tachyarrhythmias. We used single-strand conformation polymorphism (SSCP) and DNA sequence analyses to identify mutations in the cardiac sodium channel gene, SCN5A, in affected members of four LQT families. These mutations include two identical intragenic deletions and two missense mutations. These data suggest that SCN5A mutations cause LQT. The location and character of these mutations suggest that this form of LQT results from a delay in cardiac sodium channel fast inactivation or altered voltage-dependence of inactivation. ...

Background: Overexpression of the human epidermal growth factor receptor (HER) 2 is associated with poor prognosis and shortened survival in breast cancer patients. HER2 is a potent activator of several signaling pathways that support cell survival, proliferation and metabolism. In HER2- positive breast cancer there are most likely unexplored proteins that act directly or indirectly downstream of well established pathways and take part in tumor development and treatment response.. Methods: In order to identify novel copy number variations (CNVs) in HER2-positive breast cancer whole-genome single nucleotide polymorphism (SNP) arrays were used. A PCR-based loss of heterozygosis (LOH) assay was conducted to verify presence of deletion in HER2-positive breast cancer cases but also in HER2 negative breast cancers, cervical cancers and lung cancers. Screening for mutations was performed using single-strand conformation polymorphism (SSCP) followed by PCR sequencing. Protein expression was evaluated ...

TY - JOUR. T1 - Identification of seven novel mutations in LH β-subunit gene by SSCP. AU - Roy, Ashim C.. AU - Liao, Wu Xiang. AU - Chen, Ying. AU - Arulkumaran, Sabaratnam. AU - Ratnam, Shan S.. PY - 1996. Y1 - 1996. N2 - Seven new point mutations have been identified from LH β-subunit gene by PCR-mediated SSCP, and sequencing. One mutation was found changing amino acid from Gln102 to Ser102. The remaining six mutations, which did not change the codings, were in complete linkage disequilibrium. SSCP can be used in the diagnosis of LH-related disorders.. AB - Seven new point mutations have been identified from LH β-subunit gene by PCR-mediated SSCP, and sequencing. One mutation was found changing amino acid from Gln102 to Ser102. The remaining six mutations, which did not change the codings, were in complete linkage disequilibrium. SSCP can be used in the diagnosis of LH-related disorders.. KW - Luteinizing hormone. KW - Mutation. KW - Polymerase chain reaction. KW - Restriction enzyme ...

Figure 2. Mutation analysis of the MYO7A gene in the family. A: Sequencing chromatograms of PCR products of exon 22 from a normal individual and the affected individual IV-4; insertion of an A residue is marked by an arrow. B: Partial nucleotide and amino acid sequences of wild type (normal) and mutant alleles; insertion site of an A residue is marked above by a pipe (,) in the wild type allele; a missense run of 18 amino acids are shown in purple in the mutant protein; premature stop codon in exon 23 is shown in red. C: PCR-SSCP analysis of all 13 individuals from the family, note all five affected individuals (IV-1, IV-2, IV-3, IV-4, and IV-6) are homozygous for the mutant allele (M) and their parents (II-1, III-1, III-2, and III-3) are heterozygous for the mutant and the wild type (W) alleles. D: Schematic representation of mutant (above) and normal MYO7A (below) proteins. Note the truncation of MYO7A protein in affected individuals. The normal protein has a head domain with an ATP binding ...

PubMed journal article: p53 gene abnormalities are closely related to hepatoviral infections and occur at a late stage of hepatocarcinogenesis. Download Prime PubMed App to iPhone, iPad, or Android

摘要 试验旨在研究DQB2基因外显子2多态性与哈萨克羊布鲁氏菌病易感性的相关性。通过PCR-SSCP技术对146只布鲁氏菌阴性哈萨克羊血液样本和28只布鲁氏菌阳性哈萨克羊血液样本中的白细胞表面抗原DQB2基因外显子2的多态性进行研究，挑取不同的等位基因进行克隆测序，经卡方检验分析每个SNP位点的基因频率、基因型频率及其多态性与布鲁氏菌病易感性的相关性，应用生物信息学软件分析与哈萨克羊布鲁氏菌病易感性相关的不同等位基因的mRNA二级结构及蛋白质的二级结构、三级结构和抗原表位。结果发现，在270 ...