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Pathology Oncology Research : POR Oct 2016Frameshift mutation of genes containing mononucleotide repeats is a feature of gastric (GC) and colorectal cancers (CRC) with microsatellite instability (MSI). In the...
Frameshift mutation of genes containing mononucleotide repeats is a feature of gastric (GC) and colorectal cancers (CRC) with microsatellite instability (MSI). In the public genome database, we found that human HSPA4 gene encoding a heats hock protein 70 protein (HSP70-4) and MED13 gene had mononucleotide repeats in the coding sequences that could be targets for frameshift mutation in cancers with MSI. HSP70-4 is a member of HSP70 that is known to play a role in cell survival. MED13 is a member of MED genome-wide transcription regulators that function as a regulator for diverse biological processes. In this study, we analyzed the mutations in 79 GCs and 124 CRCs including high MSI (MSI-H) and microsatellite stable/low MSI (MSS/MSI-L) cases by single-strand conformation polymorphism analysis and DNA sequencing. We found frameshift mutations of HSPA4 gene in two cancers (one GC and one CRC) and MED13 gene in the other two cancers (one GC and one CRC). The frameshift mutations were deletions of one base (c.2396delA (p.Asn799MetfsX50)) in HSPA4 and (c.2175delA (p.Lys725AsnfsX4)) in MED13. Each of HSPA4 and MED13 mutations were detected in GC with MSI-H (1/34: 2.9 %) and CRC with MSI-H (1/79: 1.3 %), but not in those with MSS. Our data show that unconventional HSPA4 and MED13 genes harbored frameshift mutations in GC and CRC with MSI. These mutations might possibly inactivate their functions and could be a feature of GC and CRC with MSI-H.
Topics: Colorectal Neoplasms; DNA, Neoplasm; Frameshift Mutation; HSP110 Heat-Shock Proteins; Humans; Mediator Complex; Microsatellite Instability; Polymorphism, Single-Stranded Conformational; Stomach Neoplasms
PubMed: 27129500
DOI: 10.1007/s12253-016-0070-9 -
Indian Journal of Medical Microbiology 2009Because of the emergence of multidrug-resistant tuberculosis in recent times, the rapid detection of resistance to the first-line anti-tuberculosis drug rifampicin was...
PURPOSE
Because of the emergence of multidrug-resistant tuberculosis in recent times, the rapid detection of resistance to the first-line anti-tuberculosis drug rifampicin was felt worldwide. Accordingly, this study was conducted to evaluate the diagnostic potential of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) for checking its utility as a rapid screening test for determination of rifampicin drug resistance.
MATERIALS AND METHODS
A total of 34 isolates of Mycobacterium tuberculosis (M. tuberculosis) (22 rifampicin resistant, 11 rifampicin sensitive and one control H37Rv) strains were analysed by PCR-SSCP and DNA sequencing within the 157-bp region of the rpo B gene (Ala 500-Val 550).
RESULTS
Rifampicin resistance was detected successfully by PCR-SSCP in 20/22(90.90%) of rifampicin-resistant strains showing a total of nine different mutations in seven codon positions: codon 513 (CAA-->CCA), 516 (GAC-->GTC), 507 (GGC-->GAC), 526 (CAC-->GAC, TAC), 531 (TCG-->TTG, TGG), 522 (TCG-->TGG) and 533 (GTG-->CCG). Two rifampicin-resistant strains showed an identical PCR-SSCP pattern with the wild type H37Rv; 77.27% rifampicin-resistant strains showed a single point mutation and 9.09% had no mutation. Three rifampicin-resistant strains showed characteristic double mutations at codon positions 526 and 531. Sensitivity and specificity were calculated as 90.90% and 100%.
CONCLUSIONS
Rifampicin-resistant genotypes were mainly found in codon positions 516, 526 and 531. PCR-SSCP seems to be an efficacious method of predicting rifampicin resistance and substantially reduces the time required for susceptibility testing from 4 to 6 weeks to a few weeks.
Topics: Antitubercular Agents; Bacterial Proteins; DNA-Directed RNA Polymerases; Drug Resistance, Bacterial; Humans; Microbial Sensitivity Tests; Mycobacterium tuberculosis; Point Mutation; Polymorphism, Single-Stranded Conformational; Rifampin; Sensitivity and Specificity; Sequence Analysis
PubMed: 19584503
DOI: 10.4103/0255-0857.45364 -
American Journal of Human Genetics Feb 1998Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by tumors of the parathyroids, pancreatic islets, and anterior pituitary. The...
Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by tumors of the parathyroids, pancreatic islets, and anterior pituitary. The MEN1 gene, on chromosome 11q13, has recently been cloned, and mutations have been identified. We have characterized such MEN1 mutations, assessed the reliability of SSCP analysis for the detection of these mutations, and estimated the age-related penetrance for MEN1. Sixty-three unrelated MEN1 kindreds (195 affected and 396 unaffected members) were investigated for mutations in the 2,790-bp coding region and splice sites, by SSCP and DNA sequence analysis. We identified 47 mutations (12 nonsense mutations, 21 deletions, 7 insertions, 1 donor splice-site mutation, and 6 missense mutations), that were scattered throughout the coding region, together with six polymorphisms that had heterozygosity frequencies of 2%-44%. More than 10% of the mutations arose de novo, and four mutation hot spots accounted for >25% of the mutations. SSCP was found to be a sensitive and specific mutational screening method that detected >85% of the mutations. Two hundred and one MEN1 mutant-gene carriers (155 affected and 46 unaffected) were identified, and these helped to define the age-related penetrance of MEN1 as 7%, 52%, 87%, 98%, 99%, and 100% at 10, 20, 30, 40, 50, and 60 years of age, respectively. These results provide the basis for a molecular-genetic screening approach that will supplement the clinical evaluation and genetic counseling of members of MEN1 families.
Topics: Adolescent; Adult; Age Factors; Aged; Alternative Splicing; Amino Acid Sequence; Base Sequence; Child; Child, Preschool; Chromosome Mapping; Chromosomes, Human, Pair 11; DNA Transposable Elements; Exons; Female; Humans; Male; Microsatellite Repeats; Middle Aged; Multiple Endocrine Neoplasia Type 1; Mutation; Neoplasm Proteins; Pedigree; Point Mutation; Polymorphism, Genetic; Polymorphism, Single-Stranded Conformational; Proto-Oncogene Proteins; Sequence Deletion
PubMed: 9463336
DOI: 10.1086/301729 -
The Journal of Molecular Diagnostics :... Aug 2001The human leukocyte antigens (HLA) encoded by genes within the major histocompatibility complex display an impressive degree of polymorphism. This variability is... (Review)
Review
The human leukocyte antigens (HLA) encoded by genes within the major histocompatibility complex display an impressive degree of polymorphism. This variability is apparently maintained in human populations through the need to successfully display a wide range of processed foreign peptides to the T cell antigen receptor. The large number of alleles at the Class I and Class II loci pose a significant problem for molecular diagnosis. Knowledge of allele groups and specific alleles present in individuals has important implications in organ and stem cell transplantation and in disease association studies. Histocompatibility laboratories have transformed themselves during the past decade as they have adapted the techniques of molecular diagnostics to the challenge of identifying HLA alleles.
Topics: Alleles; Amino Acid Sequence; Exons; Genes, MHC Class I; Genes, MHC Class II; HLA Antigens; Humans; Immunophenotyping; Models, Genetic; Molecular Sequence Data; Polymorphism, Genetic; Polymorphism, Single-Stranded Conformational; Sequence Analysis, DNA; Sequence Homology, Amino Acid
PubMed: 11486048
DOI: 10.1016/S1525-1578(10)60658-7 -
FEMS Immunology and Medical Microbiology Sep 1998The study of the genetic heterogeneity of P. carinii is complicated by the lack of an in vitro culture system, as well as by the likely occurrence of co-infections with... (Review)
Review
The study of the genetic heterogeneity of P. carinii is complicated by the lack of an in vitro culture system, as well as by the likely occurrence of co-infections with several special forms or types in a single host. Karyotyping and multilocus enzyme electrophoresis are useful for studies at the evolutionary level. However, these methods require a large number of cells, which prevents their use for the special form infecting humans. DNA sequence analysis of genomic regions is useful to study P. carinii diversity, both at the evolutionary and epidemiological levels. To type the special form specific to humans, several methods are currently used to detect polymorphism in PCR products of polymorphic regions of the genome: DNA sequencing, type-specific hybridisations, and single-strand conformation polymorphism. All these methods still need evaluation. The frequency of potential co-infections in humans determined by these various methods is different. The differences could be due to methodological problems or to real variations between patient populations, geographical locations and/or prophylaxis regimens. In the future, elucidating the population structure of P. carinii and the frequency of potential co-infections is going to be crucial for a better understanding of its epidemiology, and thus for a better prevention of P. carinii pneumonia in humans.
Topics: DNA, Fungal; Electrophoresis; Genetic Heterogeneity; Karyotyping; Mycological Typing Techniques; Nucleic Acid Hybridization; Pneumocystis; Polymorphism, Single-Stranded Conformational; Sequence Analysis, DNA
PubMed: 9792058
DOI: 10.1111/j.1574-695X.1998.tb01184.x -
Journal of Applied Microbiology 2005To examine the utility of polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis to differentiate epidemic and nonepidemic Vibrio...
Molecular typing of epidemic and nonepidemic Vibrio cholerae isolates and differentiation of V. cholerae and V. mimicus isolates by PCR-single-strand conformation polymorphism analysis.
AIMS
To examine the utility of polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis to differentiate epidemic and nonepidemic Vibrio cholerae isolates as well as to differentiate V. cholerae and Vibrio mimicus isolates.
METHODS AND RESULTS
By both PCR-restriction fragment length polymorphism (RFLP) and PCR-SSCP analysis of groEL-I on chromosome 1 and groEL-II on chromosome 2, V. cholerae isolates gave distinct profiles compared with V. mimicus isolates. In addition, PCR-SSCP analysis of groEL-I and groEL-II could differentiate between V. cholerae epidemic and nonepidemic isolates. Interestingly, the relationships among strains based on groEL-I from chromosome 1 and groEL-II from chromosome 2 were congruent with each other, highlighting the conserved evolutionary history of both chromosomes in this species.
CONCLUSIONS
PCR-SSCP is a powerful typing technique, which has the ability to differentiate V. cholerae and V. mimicus isolates. The epidemic V. cholerae O1/O139 serogroup isolates represent a clonal complex distinct from non-O1/non-O139 isolates that can be identified by PCR-SSCP analysis.
SIGNIFICANCE AND IMPACT OF THE STUDY
This study highlights the effectiveness of using reliable molecular typing methods and in particular PCR-SSCP, to identify genetic variation among V. cholerae and V. mimicus isolates.
Topics: Bacteriological Techniques; Base Sequence; Chaperonin 60; Cholera; Disease Outbreaks; Genes, Bacterial; Humans; Molecular Sequence Data; Polymorphism, Single-Stranded Conformational; Sequence Alignment; Vibrio cholerae; Vibrio mimicus; Water Microbiology
PubMed: 15715856
DOI: 10.1111/j.1365-2672.2004.02451.x -
Bioscience, Biotechnology, and... 2013Molecular diversity within six viroid species and different molecular variants, in each species infecting fruit trees was first estimated by the single-strand...
Molecular diversity within six viroid species and different molecular variants, in each species infecting fruit trees was first estimated by the single-strand conformation polymorphism (SSCP) technique and then by direct sequencing analysis. The different variants studied are to three Australian grapevine viroids(AGVd), four citrus dwarfing viroids (CDVd), eleven grapevine yellow speckle viroids type-1 (GYSVd-1), four hop stunt viroids (HSVd), seven peach latent mosaic viroids (PLMVd), and eight pear blister canker viroids (PBCVd). Polyacrylamide gel electrophoresis (PAGE) conditions were compared and optimized to improve the sensitivity of the existing SSCP parameters. The relationships among the various SSCP profiles observed and the variation in nucleotide sequences was studied. The results indicate that the variations of some parameters of electrophoresis for each species allowed higher resolution and hence detection of single nucleotide variations among clones initially clustered into the same group.
Topics: Base Sequence; Citrus; Electrophoresis, Polyacrylamide Gel; Humulus; Molecular Sequence Data; Nucleic Acid Conformation; Plant Diseases; Polymorphism, Single-Stranded Conformational; Prunus; Pyrus; RNA, Viral; Sequence Analysis, DNA; Viroids; Vitis
PubMed: 23291763
DOI: 10.1271/bbb.120714 -
Molecular Biology Reports Apr 2012The objective of this research was to detect bovine GDF10 gene polymorphism and analyze its association with body measurement traits (BMT) of animals sampled from 6...
The objective of this research was to detect bovine GDF10 gene polymorphism and analyze its association with body measurement traits (BMT) of animals sampled from 6 different Chinese indigenous cattle populations. The populations included Xuelong (Xl), Luxi (Lx), Qinchuan (Qc), Jiaxian red (Jx), Xianang (Xn) and Nanyang (Ny). Blood samples were taken from a total of 417 female animals stratified into age categories of 12-36 months. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was employed to find out GDF10 single polymorphism nucleotide (SNPs) and explore their possible association with BMT. Sequence analysis of GDF10 gene revealed 3 SNPs in total: 1 in exon1 (G142A) and 2 in exon3 (A11471G, and T12495C). G142A and T12495C SNPs are both synonymous mutation. They showed 2 genotypes namely respectively (GG, GA) and (PP and PB). A11471G SNP is a missense mutation leading to the change of Alanine to Threonine amino acid. It showed three genotypes namely AA, BB and AB. Analysis of association of polymorphism with body measurement traits at the three locus showed that there were significant effects on BMT in Qc, Jx and Ny cattle population. These results suggest that the GDF10 gene might have potential effects on body measurement traits in the above mentioned cattle populations and could be used for marker-assisted selection.
Topics: Animals; Base Sequence; Biometry; Cattle; Chi-Square Distribution; China; Electrophoresis, Agar Gel; Exons; Female; Gene Frequency; Genetic Association Studies; Genetic Loci; Genetics, Population; Genotype; Growth Differentiation Factor 10; Least-Squares Analysis; Molecular Sequence Data; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Polymorphism, Single-Stranded Conformational; Quantitative Trait, Heritable
PubMed: 21805344
DOI: 10.1007/s11033-011-1188-1 -
World Journal of Gastroenterology Apr 2004To analyze the characterization of T-cell receptor-gamma (TCR-gamma) gene rearrangement in the gastrointestinal lymphomas and evaluate the value of PCR-SSCP analysis in...
AIM
To analyze the characterization of T-cell receptor-gamma (TCR-gamma) gene rearrangement in the gastrointestinal lymphomas and evaluate the value of PCR-SSCP analysis in gastrointestinal lymphomas investigation.
METHODS
TCR-gamma gene rearrangement segments of gastrointestinal lymphomas were cloned and sequenced. Single clone plasmid and mixed clone plasmids were subsequently submitted to PCR-SSCP analysis to investigate the relationship between the number of amplified clones and band patterns of the amplified products. The PCR products of TCR-gamma gene rearrangement of 40 gastrointestinal lymphomas were electrophoresed on agarose gels and the positive cases on agarose gels were studied by SSCP analysis.
RESULTS
The sequencing showed that TCR-gamma gene rearrangement of the gastrointestinal lymphomas included functional gene and pseudogene with extensive variety in the junctional regions. In SSCP analysis, the number of the single-stranded bands was about two times of the number of amplified clones, and double-stranded band became broad with the increased number of the amplified clones. Thirteen of the 25 B-cell gastrointestinal lymphomas and 14 of the 15 gastrointestinal T-cell lymphomas were positive detected on agarose gel electrophoresis. Of the positive cases detected by SSCP analysis, 3 B-cell lymphomas and 13 T-cell lymphomas showed positive bands. The other cases showed only smears. The rearranged pattern included 13 monoallelic gene rearrangements and 3 biallelic or oligoclonal gene rearrangements.
CONCLUSION
The pattern of TCR-gamma gene rearrangement in gastrointestinal lymphomas are similar to that of the nodular lymphomas. PCR-SSCP analysis for TCR-gamma gene rearrangement can be applied both for adjuvant diagnosis of gastrointestinal lymphomas and analysis of the gene rearrangement pattern. The ratio of TCR-gamma gene rearrangements occurred in T-cell gastrointestinal lymphomas is significantly higher than that in B-cell gastrointestinal lymphomas. The gene rearrangement pattern involves monoallelic and biallelic (or oligoclonal) gene rearrangement.
Topics: Base Sequence; Gastrointestinal Neoplasms; Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor; Humans; Lymphoma; Molecular Sequence Data; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational
PubMed: 15052681
DOI: 10.3748/wjg.v10.i7.995 -
Microbiology (Reading, England) Mar 2003The 16S-23S rRNA internal transcribed spacer (ITS) region of several Streptococcus thermophilus strains and some related dairy streptococci, S. macedonicus, S....
16S-23S rRNA intergenic spacer region sequence variation in Streptococcus thermophilus and related dairy streptococci and development of a multiplex ITS-SSCP analysis for their identification.
The 16S-23S rRNA internal transcribed spacer (ITS) region of several Streptococcus thermophilus strains and some related dairy streptococci, S. macedonicus, S. salivarius and S. bovis, was analysed by sequence analysis. All the Streptococcus species were easily discriminated on the basis of sequence variations principally located upstream and downstream of the region encompassing the double-stranded processing sites and the tRNA(Ala) gene. Comparison between tRNA(Ala) gene sequences highlighted a high level of sequence conservation among the Streptococcus species investigated despite their belonging to separated phylogenetic clusters, i.e. the S. salivarius and S. bovis rRNA groups. A low but significant degree of variability was detected among the S. thermophilus strains, allowing the identification of four different ITS sequences. Similarity analysis of the ITS sequences showed that the Streptococcus species were clustered in two main branches, one containing S. macedonicus and S. bovis strains, and one containing S. thermophilus and S. salivarius strains. With the aim of developing a rapid tool for the identification of the dairy streptococci species a multiplex ITS-SSCP analysis of two discrete regions within the ITS locus was carried out.
Topics: Bacterial Typing Techniques; DNA, Ribosomal Spacer; Dairy Products; Dairying; Genetic Variation; Hot Temperature; Polymorphism, Single-Stranded Conformational; RNA, Ribosomal, 16S; RNA, Ribosomal, 23S; Species Specificity; Streptococcus
PubMed: 12634348
DOI: 10.1099/mic.0.25925-0