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At least 109 records · Page 6Linked to original sources
PubMed1993

Single-strand conformation polymorphism analysis is a rapid and effective method for the identification of mutations and polymorphisms in the gene for glycoprotein IIIa.

Glanzmann thrombasthenia (GT) is the most common inherited disorder of platelets. Most of the molecular defects previously identified in GT have been caused by point (or other small) mutations in the genes for glycoprotein (GP) IIb or GPIIIa. We have used single-strand conformation polymorphism (SSCP) analysis to rapidly identify single-base changes in the GPIIIa gene. Using genomic DNA from normal individuals and patients with GT, each GPIIIa exon and a short stretch of flanking intronic sequence was amplified, heat-denatured, and separated in nondenaturing acrylamide gels. Only those fragments with an abnormal migration pattern were isolated and the nucleotide sequence determined. Using SSCP, we detected the polymorphism in the HPA-1 (P1A) system and all three known silent polymorphisms in the GPIIIa gene. Screening 14 GPIIIa exons from 5 patients with GT, one mutant allele was identified. The nucleotide sequence of the abnormal 240-bp SSCP fragment was determined and a G-->A substitution in the splice donor site of exon iv was identified. Analysis of platelet RNA resulting from this mutation showed two mRNA species: one contained a deletion of exon iv, whereas the other had a 27-bp addition to exon iv due to the use of a cryptic splice site in the downstream intron. Single-base substitutions are the most common mutation in GT and often result in abnormal mRNA splicing. SSCP is a rapid and sensitive technique for identifying mutations or polymorphisms in the GPIIIa gene.

Base Sequence↗
PubMed1988

Human T cells recognize polymorphic and non-polymorphic regions of the Plasmodium falciparum circumsporozoite protein.

In order to characterize T cell epitopes in the Plasmodium falciparum circumsporozoite (CS) protein sequence, we isolated T cell clones, from non-immune donors, which reacted with synthetic peptides corresponding to two predicted CS protein T cell epitopes. Peptide CS.T3 (corresponding to a non-polymorphic region of the CS protein, residues 378-398) was recognized in association with either DR2 or DRw9 restriction elements. T cell clones recognizing CS.T3 also reacted with the sporozoite-derived CS protein. Peptide CS.T2 corresponds to a polymorphic region (residues 325-341) of the CS protein. Unlike the CS.T3-specific clones, the CS.T2-specific clones did not recognize the CS protein. Since the CS.T2 peptide includes residues which are polymorphic in different P. falciparum isolates, we investigated whether these residues were critical for recognition of the peptide. We show here that a single amino acid substitution at a position of the CS protein which shows genetic polymorphism affects recognition of the sequence by human T cells. The implications of these data for malaria vaccine development are discussed.

Animals↗
PubMed2000

Genetic linkage of Kozak sequence polymorphism of the platelet glycoprotein Ib alpha with human platelet antigen-2 and variable number of tandem repeats polymorphism, and its relationship with coronary artery disease.

The -5 C/T polymorphism of platelet glycoprotein (GP) Ib alpha is a major determinant of the level of GP Ib/V/IX complex surface expression. We investigated the frequency of this polymorphism among Asian populations. The gene frequencies of cytosine (C) in this polymorphism were 0.283 and 0.219 in Japanese and Korean populations respectively. The C allele is linked with human platelet antigen (HPA)-2a and smaller types of variable number of tandem repeats (VNTR). A novel allele, C-HPA-2a-D of VNTR, was found. No association was observed between these alleles and coronary artery disease in this case-control study. The clinical relevance of this polymorphism in the thrombotic status remains undetermined.

Adult↗
PubMed1995

Coxiella burnetii in polymorphic lymphocytes in tissue and blood of patients with polymorphic reticulosis.

Coxiella burnetii is a well-known causative agent of granulomatous inflammation and an inducer of morphological changes and transformation of human B lymphocytes in vitro. An association of the organism with polymorphic reticulosis (PMR), a malignant granulomatous inflammation characterized by polymorphic lymphocytes, was examined. The infection of C. burnetii was demonstrated in all cases tested, especially in polymorphic lymphocytes. Also the presence of morphologically transformed peripheral blood lymphocytes (PBLs) infected with C. burnetii was demonstrated. In cultures of blood lymphocytes, C. burnetii-infected polymorphic cells identified as B cells became immortalized in vitro. These findings implicate the role of C. burnetii in the process of PMR.

Adult↗
PubMed1998

Genotyping metabolic polymorphisms in a cohort of Caucasians and single strand conformation polymorphism analysis of point mutations in human hprt exons 7 and 8.

In genetic toxicology, the main fields of applications of the polymerase chain reaction (PCR) with subsequent electrophoretic characterization of amplificates include genotyping polymorphisms in the xenobiotic metabolism and mutant analysis. To assess the role of the individual sets of biotransformation enzymes for the internal dose resulting from xenobiotic exposure, we investigated blood samples from 69 healthy donors for the occurrence of known genetic polymorphisms in the xenobiotic metabolizing enzymes N-acetylaminotransfrase II (NAT2), glutathione-S-transferase (GST) mu and theta, and several cytochromes P450 (CYP), namely CYP1A1, CYP2E1 and CYP2A6. Using single strand conformation polymorphism (SSCP) analysis, five known single base substitutions located in the middle portion of 144 bp amplificates comprising exons 7 and 8 of the human hypoxanthine guanine phosphoribosyl transferase (hprt) cDNA, were clearly distinguished from wild type and from each other. Biomagnetic strand separation assigned the slower migrating single strand bands to the biotinylated sense strands.

Aryl Hydrocarbon Hydroxylases↗
PubMed1988

Simultaneous analysis of multiple polymorphic loci using amplified sequence polymorphisms (ASPs).

In this paper we present a systematic approach to gene mapping and genotyping based on the simultaneous analysis of multiple amplified sequence polymorphisms (ASPs). These genetic markers measure variation in DNA sequences which have been amplified by a polymerase and/or a ligase. The amplified sequence lengths are determined by appropriate choice of oligonucleotides used in the amplification reaction. We describe three classes of ASPs: restriction site polymorphisms, sequence length polymorphisms, and DNA base pair changes not associated with restriction sites. Simultaneous analysis of multiple ASPs using a modified automated DNA sequencing apparatus should be possible because amplification with oligonucleotides provides control over the fragment lengths generated. Development of an automated ASP technology is therefore the next logical step for efficient gene mapping and genotyping of individuals. With this technology, one gel would be sufficient to indicate the most probable locations of a gene and a second gel would permit the selection of the correct location while simultaneously providing a fine structure map.

Chromosome Mapping↗
PubMed1996

Intensive linkage mapping in a wasp (Bracon hebetor) and a mosquito (Aedes aegypti) with single-strand conformation polymorphism analysis of random amplified polymorphic DNA markers.

The use of random amplified polymorphic DNA from the polymerase chain reaction (RAPD-PCR) allows efficient construction of saturated linkage maps. However, when analyzed by agarose gel electrophoresis, most RAPD-PCR markers segregate as dominant alleles, reducing the amount of linkage information obtained. We describe the use of single strand conformation polymorphism (SSCP) analysis of RAPD markers to generate linkage maps in a haplodiploid parasitic wasp Bracon (Habrobracon) hebetor and a diploid mosquito. Aedes aegypti. RAPD-SSCP analysis revealed segregation of codominant alleles at markers that appeared to segregate as dominant (band presence/band absence) markers or appeared invariant on agarose gels. Our SSCP protocol uses silver staining to detect DNA fractionated on large thin polyacrylamide gels and reveals more polymorphic markers than agarose gel electrophoresis. In B. hebetor, 79 markers were mapped with 12 RAPD primers in six weeks; in A aygpti, 94 markers were mapped with 10 RAPD primers in five weeks. Forty-five percent of markers segregated as codominant loci in B. hebetor, while 11% segregated as codominant loci in A. aegypti. SSCP analysis of RAPD-PCR markers offers a rapid and inexpensive means of constructing intensive linkage maps of many species.

Aedes↗
PubMed1992

Insertion/deletion polymorphism and other restriction fragment length polymorphisms in the MCC gene.

The MCC gene is a candidate as a tumor suppressor gene for colorectal neoplasms. Further, MCC is tightly linked to the familial adenomatous polyposis (FAP) locus by linkage and physical analysis. Hence, restriction fragment length polymorphisms (RFLPs) of this gene might be very useful for presymptomatic diagnosis of individuals in families segregating mutant alleles of the APC gene. Here we report the identification of five polymorphic systems in MCC gene (both cDNA and genomic), one of which is an insertion/deletion polymorphism that is detectable by a polymerase chain reaction method. These five RFLP systems should be useful for linkage studies in FAP and for examining loss of heterozygosity at this locus in colonic polyps and tumors.

Adenomatous Polyposis Coli↗
PubMed1996

Characterization of Erwinia chrysanthemi by pectinolytic isozyme polymorphism and restriction fragment length polymorphism analysis of PCR-amplified fragments of pel genes.

Conserved regions about 420 bp long of the pelADE cluster specific to Erwinia chrysanthemi were amplified by PCR and used to differentiate 78 strains of E. chrysanthemi that were obtained from different hosts and geographical areas. No PCR products were obtained from DNA samples extracted from other pectinolytic and nonpectinolytic species and genera. The pel fragments amplified from the E. chrysanthemi strains studied were compared by performing a restriction fragment length polymorphism (RFLP) analysis. On the basis of similarity coefficients derived from the RFLP analysis, the strains were separated into 16 PCR RFLP patterns grouped in six clusters, These clusters appeared to be correlated with other infraspecific levels of E. chrysanthemi classification, such as pathovar and biovar, and occasionally with geographical origin. Moreover, the clusters correlated well with the polymorphism of pectate lyase and pectin methylesterase isoenzymes. While the pectin methylesterase profiles correlated with host monocot-dicot classification, the pectate lyase polymorphism might reflect the cell wall microdomains of the plants belonging to these classes.

Base Sequence↗
PubMed1998

Comparison of amplified ribosomal DNA restriction analysis, random amplified polymorphic DNA analysis, and amplified fragment length polymorphism fingerprinting for identification of Acinetobacter genomic species and typing of Acinetobacter baumannii.

Thirty-one strains of Acinetobacter species, including type strains of the 18 genomic species and 13 clinical isolates, were compared by amplified ribosomal DNA restriction analysis (ARDRA), random amplified polymorphic DNA analysis (RAPD), and amplified fragment length polymorphism (AFLP) fingerprinting. ARDRA, performed with five different enzymes, showed low discriminatory power for differentiating Acinetobacter at the species and strain level. The standardized commercially available RAPD kit clearly enabled the discrimination of all Acinetobacter genomic species but showed great polymorphism between isolates of Acinetobacter baumannii. AFLP fingerprinting with radioactively as well as fluorescently labelled primers showed high discriminatory power for the identification of 18 Acinetobacter genomic species and typing of 13 clinical Acinetobacter isolates. Compared to radioactive AFLP, fluorescent AFLP was technically fast and simple to perform, and it permitted analysis with an automated DNA sequencer. Fluorescent AFLP seems particularly well suited for studying the epidemiology of nosocomial infections and outbreaks caused by Acinetobacter species.

Acinetobacter↗
PubMed2000

Comparison of polymorphism of thymidine kinase gene and restriction fragment length polymorphism of genomic DNA in herpes simplex virus type 1.

The polymorphism of the thymidine kinase (TK) gene of herpes simplex virus type 1 (HSV-1) was analyzed and was compared with the restriction fragment length polymorphism (RFLP) of the whole genome to evaluate the relative efficiency of the TK gene as a potential probe for identification and discrimination of HSV-1. The effectiveness of using the polymorphism of the TK gene in classifying HSV-1 strains was comparable to that of RFLP analysis of 66 sites, suggesting that TK gene sequencing may have important applications in epidemiological studies of HSV-1.

DNA, Viral↗
PubMed1991

[DNA polymorphism in the Mongolian population. Restriction fragment length polymorphism analysis of mitochondrial DNA].

Restriction enzyme fragment patterns in the D loop and deletion-insertion polymorphism in the V noncoding region of human mitochondrial DNA (mt DNA) were analysed in Mongolian population using the polymerase chain reaction. Polymorphisms were detected and mt DNAs classified into 40 types using seven enzymes--AvaII, BamHI, CfrI131, KpnI, EcoRV, HaeIII RsaI and Asian specific deletion and insertion. The allele frequencies of the polymorphisms and gene diversity were determined. The data obtained for Mongolian population and the literature data were comparatively studied.

Alleles↗
PubMed1993

Deletion mapping of chromosome 8 in cancers of the urinary bladder using restriction fragment length polymorphisms and microsatellite polymorphisms.

We have used a combination of restriction fragment length polymorphism (RFLP) markers and highly informative microsatellite polymorphisms to map a common region of deletion on chromosome 8p in cancers of the urinary bladder. Analysis of loss of heterozygosity (LOH) using microsatellite polymorphisms was shown to be at least as sensitive as detection of RFLPs by Southern blotting. A total of 110 tumours was analysed for loss of heterozygosity (LOH) on 8p and 8q; 109 patients were informative for at least one marker on each chromosome arm and 29 tumours (26%) showed LOH of chromosome 8 markers, 26 of which (25%) showed LOH on 8p. Sixteen tumours (14%) showed LOH on 8q. Thirteen of these also had LOH on 8p. Of the 29 tumours with LOH, five had LOH at all informative loci, indicating loss of an entire copy of chromosome 8. An association was found between high tumour grade and stage and chromosome 8 LOH. Fifty-three per cent of grade 3 muscle-invasive tumours showed LOH compared with 11% of grade 1 non-invasive tumours (0.01 < P < 0.025 and 0.025 < P < 0.05 for grade and stage respectively). Deletion mapping of tumours with chromosome 8 LOH suggests the presence of a suppressor gene(s) for urothelial cancer within a region defined by the loci NEFL and PLAT (8p21-q11.2). If there is a common target for deletions in bladder and those in hepatocellular and colorectal tumours reported previously, this defines a common region of deletion at 8p21.3.

Base Sequence↗
PubMed1992

A new intragenic polymorphism detected by the single-strand conformation polymorphism (SSCP) assay in the dystrophin gene.

We have employed the single strand conformation polymorphism (SSCP) technique to examine a group of patients with Duchenne or Becker muscular dystrophy who do not contain deletions detectable by multiplex PCR or Southern/cDNA, in an attempt to identify uncommon mutations within the dystrophin gene. In SSCP analysis, a mutated sequence can be detected as a change of mobility in a nondenaturing polyacrylamide gel. During the course of this investigation, we detected and characterized a new polymorphism at the 3' end of intron 16. The G-to-T base change creates a TaqI restriction site which allows for rapid typing of the polymorphism by restriction digestion and electrophoresis of PCR amplified products. Its localization inside the 5' region of the dystrophin gene and its high heterozygosity makes it a useful and easy tool for rapid carrier and prenatal diagnosis.

Base Sequence↗
PubMed1998

Detection of a high-frequency silent polymorphism (C-->T) in the kir2.1 (KCNJ2) inwardly rectifying potassium channel gene by polymerase chain reaction and single strand conformation polymorphism.

The aim of this work was to determine the frequency of a base substitution (C-->T) identified in the Kir2.1 gene (approved gene symbol: KCNJ2; OMIM number: 600681). Polymerase chain reaction (PCR) of the area of the Kir2.1 gene containing this substitution was performed on 52 genomic DNA samples. Using single strand conformation polymorphism (SSCP) analysis, the genotype and allele frequencies were subsequently determined and the polymorphism identified in this study was verified by cycle sequencing. The data demonstrate that the C-->T nucleotide change identified corresponds to a silent polymorphism with a relatively high frequency. The deduced genotype frequencies of homozygotes and heterozygotes were: C/C: 73%; T/T: 2% and C/T: 25%. The deduced allele frequencies were C: 85.6% and T: 14.4%.

DNA↗
PubMed1992

Single-strand conformational polymorphisms (SSCP): detection of useful polymorphisms at the dystrophin locus.

We searched for DNA polymorphisms in seven amplified fragments of the dystrophin gene. Three fragments exhibited variable mobilities during nondenaturing strand-separating gel electrophoresis (SSGE). These variants were due to single base changes (three transversions and one transition). Three were intronic (upstream from exons 17, 15, and 48) and one was in exon 48. The frequencies of these sequence variants were determined in a sample of 54 normal X chromosomes of Caucasian origin. One of these DNA polymorphisms was observed in every 650 bp tested and the average heterozygosity was 0.05% per base pair (0.08% if exons were excluded). Such a detection density and the fact that single-strand conformational polymorphisms do not depend on the presence of any specific sequence makes them especially valuable as genetic markers. In the dystrophin locus this approach could allow simultaneous detection of frequent deletions.

DNA, Single-Stranded↗
PubMed1992

A single base pair polymorphism in the WT1 gene detected by single-strand conformation polymorphism analysis.

The Wilms' tumor predisposition gene, WT1, was analysed exon-by-exon in a variety of tumours using the single-strand conformation polymorphism (SSCP) technique. A consistent variation in the usual band pattern for exon 7 was detected in this survey. On sequencing, a silent mutation was noted in codon 313 resulting in an A-->G transition in an arginine codon. The A-->G transition destroys an AflIII restriction enzyme recognition site, which provides a rapid means of identifying heterozygotes at this locus. Analysis of the segregation of this polymorphism in families demonstrated a co-dominant inheritance pattern. In an analysis of 21 randomly selected individuals 25% were heterozygous at this locus, which makes this polymorphism useful in a variety of genetic analyses.

Base Sequence↗