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1.
Acta Pharmaceutica Sinica ; (12): 906-909, 2015.
Article in Chinese | WPRIM | ID: wpr-257048

ABSTRACT

β-Amyrin synthase (β-AS) genes of Glycyrrhiza uralensis from 6 different regions were analyzed by PCR-SSCP and sequenced, then the correlationship between β-AS SNP and regions of Glycyrrhiza uralensis were determined. According to the 1 coding single nucleotide polymorphism on the first exon of β-AS gene at 94 bp site, Glycyrrhiza uralensis could be divided into 3 genotypes. In these genotypes, the percentage of 94A type in genuine regions was much higher, and it had significant differences with the percentage in non-genuine regions (P < 0.001). The results of the experiment proved that different β-AS genotypes at 94 bp site from different regions may be one of the important reasons to result in the genuineness of Glycyrrhiza uralensis.


Subject(s)
Exons , Genotype , Glycyrrhiza uralensis , Classification , Genetics , Intramolecular Transferases , Genetics , Plant Proteins , Genetics , Polymorphism, Single Nucleotide , Polymorphism, Single-Stranded Conformational
3.
Rev. chil. pediatr ; 85(2): 213-221, abr. 2014. ilus, tab
Article in Spanish | LILACS | ID: lil-711583

ABSTRACT

Introducción: La Enfermedad Granulomatosa Crónica (EGC) se presenta como consecuencia de mutaciones en los genes que codifican 5 de las subunidades del sistema NADPH oxidasa humano. Su forma más común es causada por cambios en el gen CYBB que codifica gp91 phox. Objetivo: Identificar el defecto molecular que lleva a la presentación de la EGC. Caso clínico: Paciente de sexo masculino con antecedentes de enfermedad diarreica aguda y abscesos perianales recurrentes desde los 2 meses. A los 6 meses, presentó una inflamación crónica del colon y colitis bacteriana. A los 3 años tenía infecciones en las vías respiratorias inferiores y perianales. Estudio compatible con EGC. El análisis del ADNc identificó expresión anormal del ARNm, lo cual se confirmó al realizar la secuenciación. Específicamente se observó la ausencia del exón 2. Adicionalmente, los datos de la secuenciación del ADNg identificaron una alteración en el sitio aceptor de "splicing" del intrón 1, que incluye una deleción seguida de la inserción de 3 nucleótidos (c.46-14_-11delTTCT insGAA). Conclusiones: Se presenta el primer estudio molecular de un paciente con EGC por defecto de "splicing" reportado en Colombia. La definición de la mutación y su correlación con el fenotipo es importante para proveer una apropiada consejería genética al paciente y su familia.


Chronic granulomatous disease (CGD) is caused by mutations in the genes that encode five of the subunits of the human NADPH oxidase. The most common form is caused by mutations in CYBB, the human gene encoding gp 91 phox. Objective: To identify the molecular defects causing CGD. Case report: A male patient with a history of acute diarrhea and recurrent perianal abscess since two months old. At 6 months, the patient presented a chronic inflammatory disease of the colon and bacterial colitis. After three years, he developed infections in the lower and perianal respiratory tract. The cDNA analysis identified abnormal mRNA expression, which was confirmed by sequencing. Specifically the exclusion of exon 2 was observed. Additionally, gDNA sequencing identified an alteration in the acceptor splice site of intron 1, including a deletion followed by insertion of three nucleotides (c.46-14_-11delTTCT insGAA). Conclusions: The first molecular study of a patient with CGD due to splicing pattern change, reported in Colombia, is presented. The definition of the mutation and its correlation with the phenotype is essential to provide appropriate genetic counseling to patients and their families.


Subject(s)
Humans , Male , Infant , Chromosomes, Human, X , Granulomatous Disease, Chronic/genetics , Mutation , NADPH Oxidases/genetics , DNA, Complementary/genetics , RNA, Messenger/genetics , Base Sequence , Cell Separation , Exons , Granulomatous Disease, Chronic/diagnosis , Flow Cytometry , Phenotype , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , RNA Splicing
4.
Journal of Veterinary Research. 2014; 69 (1): 49-55
in Persian | IMEMR | ID: emr-157610

ABSTRACT

Lactoferrin [Lf], an iron binding glycoprotein, has a variety of physiological roles and its foremost is antimicrobial properties. Therefore, the lactoferrin gene can be considered as a potential candidate gene for resistance to mastitis. This study was carried out to determine the haplotype frequency of the 5'-flanking region of bovine lactoferrin gene in local and Holstein cattle breeds of Iran using PCR-SSCPand DNA sequencing. Genomic DNA was isolated from 100 blood samples of two cattle breeds [50 Local, 50 Holstein]. Two new primer pairs were designed from Lf sequence to amplify a part of 5'-flanking region of the gene. The amplified fragment was screened by single strand conformation polymorphism [SSCP] and DNA sequencing. The multiple alignments were carried out for the nucleotide sequences of different SSCPpatterns. In silico analysis of identified single nucleotide polymorphisms [SNPs] within the 5'-flanking region of bovine Lf gene was screened, to identify any association on transcription factor binding affinity. Analysis of the whole samples revealed three SSCP patterns [A, B and C] for amplified fragment that C haplotype was the only variant identified in local breed samples. The bioinformatics analysis revealed that the Tto G trans version at position -602 created an AML-1 transcription binding site in combined genotype A. In -586 position, Tto C transition abolished binding site of AML-1 transcription factor. Therefore, to apply Lf gene for marker-assisted selection, additional studies are required to evaluate the functional role of these identified polymorphic sites on gene expression and somatic cell counts in cattle


Subject(s)
Animals , Mastitis, Bovine/genetics , Polymorphism, Single-Stranded Conformational , Haplotypes/genetics , Polymorphism, Single Nucleotide , Gene Expression , Transcription Factors , Cattle/genetics , Computational Biology , Genotype
5.
JCPSP-Journal of the College of Physicians and Surgeons Pakistan. 2014; 24 (10): 749-753
in English | IMEMR | ID: emr-149784

ABSTRACT

To determine the frequency of p53 gene mutation and protein expression in Oral Squamous Cell Carcinoma [OSCC] and to establish correlation between the two. Analytical study. Histopathology Department and Molecular Biology Laboratory, Armed Forces Institute of Pathology [AFIP], Rawalpindi, from May 2010 to May 2011. Thirty diagnosed cases of OSCC were selected by consecutive sampling. Seventeen were retrieved from the record files of the AFIP, and 13 fresh/frozen sections were selected from patients reporting to the Oral Surgery Department, Armed Forces Institute of Dentistry [AFID]. Gene p53 mutation was analyzed in all the cases using PCRSSCP analysis. DNA was extracted from the formalin-fixed and paraffin-embedded tissue sections and fresh/frozen sections. DNA thus extracted was amplified by polymerase chain reaction. The amplified products were denatured and finally analyzed by gel electrophoresis. Gene mutation was detected as electrophoretic mobility shift. The immunohistochemical marker p53 was applied to the same 30 cases and overexpression of protein p53 was recorded. Immunohistochemical expression of marker p53 was positive in 67% [95% Confidence Interval [CI] 48.7 - 80.9] of the cases. Mutations of the p53 gene were detected in 23% [95% CI 11.5 - 41.2] of the OSCC. No statistically significant correlation was found between p53 gene mutation and protein p53 expression [rs = - 0.057, p = 0.765]. A substantial number of patients have p53 gene mutation [23%] and protein p53 expression [67%] in oral squamous cell carcinoma [OSCC]


Subject(s)
Humans , Male , Female , Carcinoma, Squamous Cell , Genes, p53 , Mutation , Tumor Suppressor Protein p53 , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Electrophoresis , Immunohistochemistry
6.
Article in Chinese | WPRIM | ID: wpr-310922

ABSTRACT

<p><b>OBJECTIVE</b>To analyze the fungal composition in Massa Medicata Fermentata based on culture dependent method and independent PCR-SSCP technique.</p><p><b>METHOD</b>Fungi were directly isolated from Massa Medicata Fermentata samples. The obtained strains were identified according to morphology and DNA sequence. Meanwhile the total fungal DNA was extracted from Massa Medicata Fermentata samples, the cultural independent PCR-SSCP technique based on β-tubulin gene were used to identify the mycobiota.</p><p><b>RESULT</b>According to cultural method, Aspergillus flavus and Rhizopus oryzae were present in Massa Medicata Fermentata samples, while A. flavus and A. niger were present in fried Massa Medicata Fermentata samples. In contrast, 5 species were obtained by PCR-SSCP technique, A. flavus was overlapped with fungal taxa derived from culture dependent method; A. ambiguu and A. s ivoriensis were dominant with relative abundance of 57% and 35% respectively, while the relative abundance of A. flavus was as low as 4%. None species was obtained from fried Massa Medicata Fermentata samples.</p><p><b>CONCLUSION</b>PCR-SSCP based on β-tubulin gene could distinguish fungi into species, culture dependent method combined with culture independent method could better understand the fungal composition associated with Massa Medicata Fermentata fermentation.</p>


Subject(s)
Fermentation , Fungi , Medicine, Chinese Traditional , Polymerase Chain Reaction , Methods , Polymorphism, Single-Stranded Conformational , Tubulin , Genetics
7.
Article in Chinese | WPRIM | ID: wpr-291706

ABSTRACT

<p><b>OBJECTIVE</b>To identify and confirm a novel HLA allele in a Chinese Han individual.</p><p><b>METHODS</b>HLA typing was performed by polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSOP) for HLA-A, -B and -DRB1 in a registered donor of China Marrow Donor Program(CMDP). Sequencing-based typing (SBT) was carried out to further confirm the novel allele of HLA-DRB1.</p><p><b>RESULTS</b>The SSOP result showed HLA-DRB1*09:03,15GEP, but an unusual pattern that could not be defined indicated potential presence of a novel allele. The SBT result showed the novel sequence has 1nt change from its closest allele DRB1*09:01:02 at nt306 where C to T(codon 73 GCC to GCT) resulting in no amino acid change. 73A was not changed.</p><p><b>CONCLUSION</b>A novel HLA allele, HLA-DRB1*09:01:07, has been identified and named officially by WHO Nomenclature Committee for Factors of the HLA System.</p>


Subject(s)
Base Sequence , Blood Donors , HLA-DRB1 Chains , Genetics , Histocompatibility Testing , Methods , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Sequence Analysis, DNA
8.
Acta Medica Philippina ; : 17-22, 2014.
Article in English | WPRIM | ID: wpr-633713

ABSTRACT

OBJECTIVE: This study aimed to detect the presence of microsatellite (MSI) and loss of heterozygosity (LOH) of the Deleted in Colorectal Cancer (DCC) gene in normal and tumor tissues of Filipino colorectal cancer patients and examine its correlation with age, gender, tumor grade, tumor stage and site of lesion.METHODS: Paired frozen normal and tumor tissues from thirtynine (39) patients with colorectal adenocarcinoma were used by polymerase chain reaction (PCR). Single strand conformation polymorphism - polyacrylamide gel electrophoresis (SSCP - PAGE) was used to determine MSI and restriction fragment length polymorphism (RFLP) was used to study LOH.RESULTS: Based on our data, out of the 39 patients, 10 showed LOH of the DCC gene using the LOH markers VNTR, M2 and M3, while no MSI was detected in the samples using the MSI markers BAT25 and BAT26. Correlation with clinicopathological characteristics showed that there is significance for the site of lesion. The LOH has correclation with tumor samples from the colon but not with those from the rectum.CONCLUSION: Preliminary screening for MSI and LOH of the DCC gene shows that occurrences of colorectal cancer among Filipino patients can be correlated with LOH of the DCC gene with colorectal cancer in a Filipino sample population.


Subject(s)
Humans , Male , Female , Aged , Middle Aged , Adult , Genes, DCC , Polymorphism, Single-Stranded Conformational , Colorectal Neoplasms , Adenocarcinoma , Loss of Heterozygosity
9.
Biomédica (Bogotá) ; 33(1): 53-61, ene.-mar. 2013. ilus, tab
Article in English | LILACS | ID: lil-675132

ABSTRACT

Introduction. Retinoblastoma is a childhood cancer of the retina originated by altered or null retinoblastoma protein (pRb) expression. Genetic alterations in both RB1 alleles in the retinal cells are required for the development of retinoblastoma. In the sporadic form, non-hereditary RB1 gene mutations take place in a single retinoblast cell, and are therefore only present in tumor DNA (somatic mutations). Sporadic retinoblastoma is primarily unilateral, lacks family history and has no risk of transmission to descendants. Genetic tests for detection of RB1 mutation has improved the identification of carriers and facilitated accurate genetic counseling. Objective. To identify mutations in the RB1 gene in Colombian patients with sporadic retinoblastoma by PCR-SSCP followed by sequence. Materials and methods. Four patients with sporadic retinoblastoma were analyzed by PCR-SSCP, followed by DNA sequencing to identify variations in the RB1 gene. Results. We identified five variations in RB1 gene: three new mutations (one germline and two somatic mutations), one new polymorphism and one already reported somatic mutation. Four mutations were found in three patients with unilateral retinoblastoma and one mutation was found in a patient with bilateral retinoblastoma. One of these was a germline mutation in a sporadic unilateral retinoblastoma that was not present in the parents or three siblings analyzed. Conclusions. Our results emphasize the importance of identifying mutations for genetic counseling and clinical management of sporadic retinoblastoma patients. Description of a new RB1 gene variant is interesting since there have been a small number of polymorphisms reported for this gene.


Introducción. El retinoblastoma es un cáncer pediátrico de la retina originado por la expresión alterada o ausente de la proteína del retinoblastoma (pRb). Se requiere la alteración genética de ambos alelos RB1 en las células de la retina para el desarrollo del retinoblastoma. En la forma esporádica, las mutaciones no hereditarias del gen RB1 ocurren en un solo retinoblasto y están presentes sólo en el ADN del tumor (mutaciones somáticas). El retinoblastoma esporádico es generalmente unilateral, no tiene historia familiar y no tiene riesgo de transmisión a la descendencia. Las pruebas genéticas para la detección de mutaciones en RB1 han mejorado la identificación de portadores y han facilitado la precisión de la asesoría genética. Objetivo. Detectar mutaciones en el gen RB1 en pacientes colombianos con retinoblastoma esporádico mediante PCR-SSCP seguido de secuenciación. Materiales y métodos. Se analizaron cuatro pacientes con retinoblastoma esporádico para la detección de variaciones en el gen RB1 mediante PCR-SSCP, seguida de secuenciación. Resultados. Se identificaron cinco variaciones del gen RB1 : tres mutaciones nuevas (una de línea germinal y dos somáticas), un polimorfismo nuevo y una mutación somática ya reportada. Las cuatro mutaciones se encontraron en tres pacientes con retinoblastoma unilateral y uno con bilateral. La mutación germinal se detectó en un paciente con compromiso unilateral y no se encontró en los padres ni en los tres hermanos analizados. Conclusión. Estos resultados enfatizan la importancia, para asesoría genética y manejo clínico, de identificar mutaciones del gen RB1 en pacientes con retinoblastoma esporádico. La descripción de una nueva variante en RB1 es interesante, dado el muy bajo número de polimorfismos reportados para este gen.


Subject(s)
Child, Preschool , Female , Humans , Infant , Male , Eye Neoplasms/genetics , Genes, Retinoblastoma , Mutation , Retinoblastoma/genetics , DNA Mutational Analysis , DNA, Neoplasm/analysis , DNA, Neoplasm/blood , DNA, Neoplasm/genetics , Eye Neoplasms/blood , Frameshift Mutation , Germ-Line Mutation , Neoplasms, Multiple Primary/blood , Neoplasms, Multiple Primary/genetics , Pedigree , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Retinoblastoma/blood , Sequence Analysis, DNA
10.
Chinese Journal of Biotechnology ; (12): 904-913, 2013.
Article in Chinese | WPRIM | ID: wpr-233189

ABSTRACT

We have assessed the relationships between immune trait (antibody titers of Sheep red blood cell, SRBC; Avian influenza, AI; Newcastle disease, ND) and varieties of MHC B-LBHII Gene in local chicken breeds (Wenshang Barred chicken, LH; Laiwu Black chicken, LWH; and Jining Bairi chicken, BR). We selected 300 chickens randomly from the three indigenous chicken populations. The variations of MHC B-L BII gene were detected by directly DNA sequencing and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). The results indicated that there were about 19-22 nucleotide mutations in the three local breeds, which could affect 16-18 amino acid variations. Another results indicated that there was significantly relationship between seven to eight SNPs of the MHC B-LBII region and some immune traits (P < 0.05 or P < 0.01). Both locus G97A and locus T138A were found in the three species, which were significantly related to the antibodies of SRBC, ND and AI antibody titers (P < 0.05). Among them, the locus G97A was significantly associated with ND antibody titers (P < 0.05) in BR chicken, with SRBC antibody titers (P < 0.05) in LWH chicken, and with H9 antibody titers (P < 0.05) in LH chicken. Furthermore, locus T138A was significantly associated with H9 antibody titers in BR and LH chickens (P < 0.05). All those results suggest relationships among the different varieties of MHC B-LBII and immune traits in the three local breeds.


Subject(s)
Animals , Base Sequence , Breeding , Chickens , Genetics , Allergy and Immunology , Major Histocompatibility Complex , Genetics , Polymerase Chain Reaction , Polymorphism, Single Nucleotide , Polymorphism, Single-Stranded Conformational , Sequence Analysis, DNA
11.
Rev. Soc. Bras. Med. Trop ; 45(1): 95-99, Jan.-Feb. 2012. ilus
Article in English | LILACS | ID: lil-614916

ABSTRACT

INTRODUCTION: The aim of this work was to evaluate the prevalence of Mycobacterium tuberculosis (MT) strains with mutations that could result in resistance to the main drugs used in treatment in a region with one of the highest numbers of tuberculosis (TB) cases in southern Brazil. METHODS: Deoxyribonucleic acid (DNA) from 120 sputum samples from different patients suspicious of pulmonary tuberculosis who attended the Municipal Public Laboratory for Mycobacterium sp. diagnosis was directly amplified and analyzed by PCR-SSCP. The DNA was amplified in known hotspot mutation regions of the genes rpoB, ahpC, embB, katG, inhA, and pncA. RESULTS: The percentage of samples positive by culture was 9.2 percent (11/120); 5 percent (6/120) were positive by bacilloscopy and MT-PCR, and DNA fragments of the aforementioned resistance genes could be amplified from seven (7) of the eleven (11) samples with positive results, either by culture or PCR/bacilloscopy. All presented a SSCP pattern similar to a native, nonresistant genotype, with the ATCC strain 25177 as control, except for one sample (0.01 percent), which presented a SSCP profile demonstrating mutation at the embB gene. CONCLUSIONS: These results are consistent with the empirical observations by physicians treating TB patients in our region of a low occurrence of cases that are refractory to conventional treatment schemes, in contrast to other parts of the country. Continued surveillance, especially molecular, is essential to detect and monitor the outbreak of MT-resistant strains.


INTRODUÇÃO: O objetivo deste trabalho foi avaliar a prevalência de cepas de Mycobacterium tuberculosis (MT) com mutações que podem resultar em resistência às principais drogas utilizadas no tratamento em uma das regiões com o maior número de casos de tuberculose (TB) no Sul do Brasil. MÉTODOS: O ácido desoxiribonucleico (DNA) de 120 amostras de escarro de diferentes pacientes com suspeita de TB pulmonar que procuraram o serviço público de saúde do município sede da região para o diagnóstico de MT foi diretamente amplificado e analisado por PCR-SSCP. Foram amplificadas regiões conhecidas onde ocorrem a maioria das mutações nos genes rpoB, ahpC, embB, katG, inhA, and pncA. RESULTADOS: Nove virgula dois por cento (11/120) das amostras apresentaram resultado positivo por cultura, 5 por cento (6/120) foi positiva por bacilscopia e PCR para MT, e os fragmentos dos genes mencionados puderam ser amplificados em sete (7) dos onze (11) casos com resultado positivo, seja por cultura ou PCR/baciloscopia. Todos estes casos apresentaram um padrão de SSCP similar ao genótipo nativo, não resistente, por comparação com a cepa controle ATCC 25177, com exceção de uma amostra (0,01 por cento), que apresentou um padrão de SSCP mutante no gene embB. CONCLUSÕES: Estes resultados são consistentes com as observações empíricas por parte dos clínicos que tratam os pacientes com TB na região, de uma baixa ocorrência de casos refratários ao tratamento convencional, em contraste com outras partes do país. Porém, a vigilância contínua, especialmente molecular, é essencial para identificar e monitorar o aparecimento de cepas de MT resistentes.


Subject(s)
Humans , Antitubercular Agents/pharmacology , Mutation/genetics , Mycobacterium tuberculosis/drug effects , Sputum/microbiology , Tuberculosis, Pulmonary/drug therapy , Microbial Sensitivity Tests , Mycobacterium tuberculosis/genetics , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Tuberculosis, Pulmonary/diagnosis , Tuberculosis, Pulmonary/microbiology
12.
Tehran University Medical Journal [TUMJ]. 2012; 69 (10): 619-623
in Persian | IMEMR | ID: emr-114032

ABSTRACT

Gastric cancer is one of the most common diseases of digestive system with a low 5-year survival rate and metastasis is the main cause of death. Multi-factors, such as changes in molecular pathways and deregulation of cells are involved in the disease development. Epidermal growth factor receptor pathway [EGFR] which is associated with cell proliferation and survival can influence cancer development. EGFR function is governed by its genetic polymorphism; thus, we aimed to study the tyrosine kinase domain gene mutations of the receptor in patients with gastric cancer. In this experimental study, 123 subjects [83 patients with gastric cancer and 40 normal subjects] were investigated in north of Iran for EGFR gene polymorphisms during 1 year. Genomic DNA was extracted by DNA extraction kit according to the manufacture's protocol. Polymerase chain reaction single-stranded conformation polymorphism [PCR-SSCP] and silver staining were performed for investigating EGFR gene polymorphisms. The participants included 72 men and 44 women. Gene polymorphism in exon 18 was present in 10% of the study population but SSCP pattern in exon 19 did not show different migrate bands neither in patients nor in normal subjects. It seems that screening for tyrosine kinas gene polymorphism of epidermal growth factor receptor in patients with gastric cancer and use of tyrosine kinas inhibitors could be useful in the prevention of disease progress and improvement of treatment process for a better quality of life in these patients


Subject(s)
Humans , Male , Female , Polymorphism, Genetic , ErbB Receptors , Stomach Neoplasms/genetics , Mutation , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational
13.
Iranian Journal of Cancer Prevention. 2012; 5 (1): 27-32
in English | IMEMR | ID: emr-117541

ABSTRACT

Acute Myeloid Leukaemia [AML] is a cancer of blood-forming cells in bone marrow. C-kit gene is a Receptor Tyrosine Kinase class III [RTK] that is expressed by early hematopoietic progenitor cells and plays an important role in hematopoietic stem cell proliferation, differentiation and survival. It is known that c-kit is a proto-oncogene and the activating c-kit mutations are likely to contribute in the development of leukaemia in humans. Exon 11 of c-Kit gene is the frequent site for mutations in different kinds of tumours. In order to determine the frequency and prevalence of exon 11 mutations in 51 AML cases, we have done polymerase chain reaction-single-strand conformational polymorphism followed by direct DNA sequencing. The c-kit mutations in exon 11 were detected in 15.68% [8/51] in AML cases. We have detected totally ten missense mutations in eight AML cases those include Lys550Asn, Tyr568Ser, Ile571Leu, Tyr578Pro, Trp582Ser and Arg588Met and novel missense mutations at codons Ile563Lys and Val569Leu. Mutations at codons Ile571Leu and Trp582Ser was found in two independent cases. The presence of c-kit mutations in our study adds to investigative spectrum of AML cases. Since the c-kit mutations are seen in other malignancies, mutations in exon 11 of the c-kit gene might be involve in pathogenesis and represent useful predictive genetic marker in AML. Further studies in larger group of cases possibly will be required to determine the prognostic implications and to investigate how these mutations are co-related to the progression and pathogenesis of AML


Subject(s)
Leukemia, Myeloid/genetics , Bone Marrow Cells , Polymorphism, Single-Stranded Conformational , Polymerase Chain Reaction , Sequence Analysis, DNA
14.
Article in Chinese | WPRIM | ID: wpr-346845

ABSTRACT

<p><b>OBJECTIVE</b>To establish a method for identifying Panax ginseng and P. quinquefolius with PCR-SSCP, on the basis of specific SNP identification sites of their ITS2 bar codes.</p><p><b>METHOD</b>ITS2 sequences of P. ginseng and P. quinquefolius recorded in GenBank were searched to conduct a comparative analysis and screen out specific SNP identification sites of their ITS2 bar codes. Based on that, the Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) method was adopted for analyzing 11 P. ginseng samples and 10 P. quinquefolius samples and verifying sequencing of their PCR products.</p><p><b>RESULT</b>The P. ginseng and P. quinquefolius samples had the same agarose mages of PCR-SSCP with the standard medicines. There were significant differences between the PCR-SSCP agarose mages of P. ginseng and P. quinquefolius, with identifical identification results between PCR-SSCP and sequencing.</p><p><b>CONCLUSION</b>Compared with the sequencing method, PCR-SSCP is so rapid and accurate that it can be used for identifying P. ginseng and P. quinquefolius medicines.</p>


Subject(s)
DNA Barcoding, Taxonomic , Methods , DNA, Plant , Genetics , DNA, Ribosomal Spacer , Genetics , Electrophoresis, Agar Gel , Panax , Classification , Genetics , Polymerase Chain Reaction , Polymorphism, Single Nucleotide , Polymorphism, Single-Stranded Conformational , Reproducibility of Results , Species Specificity
15.
Indian J Ophthalmol ; 2011 Sept; 59(5): 373-377
Article in English | IMSEAR | ID: sea-136208

ABSTRACT

Purpose: To study the susceptibilities of Aspergillus species against amphotericin B in infectious keratitis and to find out if drug resistance had any association with the molecular characteristics of the fungi. Materials and Methods: One hundred and sixty Aspergillus isolates from the corneal scrapings of patients with keratitis were tested for susceptibilities to amphotericin B by broth microdilution method. These included Aspergillus flavus (64 isolates), A. fumigatus (43) and A. niger (53). Fungal DNA was extracted by glass bead vertexing technique. Polymerase chain reaction (PCR) assay was standardized and used to amplify the 28S rRNA gene. Single-stranded conformational polymorphism (SSCP) of the PCR product was performed by the standard protocol. Results: Of the 160 isolates, 84 (52.5%) showed low minimum inhibitory concentration (MIC) values (≤ 1.56 μg/ml) and were designated as amphotercin B-sensitive. Similarly, 76 (47.5%) had high MICs (≥ 3.12 μg/ml) and were categorized as amphotericin B-resistant. MIC50 and MIC90 values ranged between 3.12-6.25 μg/ml and 3.12-12.5 μg/ml respectively. A. flavus and A. niger showed higher MIC50 and MIC90 values than A. fumigatus. The SSCP pattern exhibited three extra bands (150 bp, 200 bp and 250 bp each) in addition to the 260 bp amplicon. Strains (lanes 1 and 7) lacking the 150 bp band showed low MIC values (≤ 1.56 μg/ml). Conclusion: A. niger and A. flavus isolates had higher MICs compared to A. fumigatus, suggesting a high index of suspicion for amphotericin B resistance. PCR-SSCP was a good molecular tool to characterize Aspergillus phenotypes in fungal keratitis.


Subject(s)
Antifungal Agents/pharmacology , Aspergillosis/drug therapy , Aspergillosis/microbiology , Aspergillus/drug effects , Aspergillus/genetics , Aspergillus/isolation & purification , Cornea/microbiology , Drug Resistance, Fungal , Eye Infections, Fungal/diagnosis , Eye Infections, Fungal/microbiology , Keratitis/diagnosis , Keratitis/microbiology , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , RNA, Fungal/analysis
16.
Mem. Inst. Oswaldo Cruz ; 106(4): 502-504, June 2011. ilus
Article in English | LILACS | ID: lil-592195

ABSTRACT

Heteroduplex mobility assay, single-stranded conformation polymorphism and nucleotide sequencing were utilised to genotype human parvovirus B19 samples from Brazil and Paraguay. Ninety-seven serum samples were collected from individuals presenting with abortion or erythema infectiosum, arthropathies, severe anaemia and transient aplastic crisis; two additional skin samples were collected by biopsy. After the procedure, all clinical samples were classified as genotype 1.


Subject(s)
Female , Humans , Pregnancy , Heteroduplex Analysis/methods , Parvoviridae Infections , Polymorphism, Single-Stranded Conformational , Base Sequence , Brazil , Genotype , Molecular Sequence Data , Paraguay , Pregnancy Complications, Infectious
17.
Genet. mol. biol ; 34(3): 456-458, 2011. ilus, tab
Article in English | LILACS | ID: lil-595981

ABSTRACT

The aim was to detect the presence of polymorphisms at exons 1, 2, 3 and 4 of the Spi2 gene, and evaluate a possible association between them and recurrent airway obstruction (RAO) or inflammatory airway disease (IAD) in thoroughbred horses, through single-strand conformational-polymorphism (SSCP) screening. Although polymorphism was not detected in exons 1, 2 and 3, three alleles and six genotypes were identified in exon 4. The frequencies of allele A (0.6388) and genotype AA (0.3888) were higher in horses affected by RAO, although no association was found between polymorphism and horses with either RAO or IAD.


Subject(s)
Animals , Horses/genetics , Polymorphism, Genetic , Respiratory Tract Diseases , alpha 1-Antitrypsin , Polymorphism, Single-Stranded Conformational , Serpins
18.
Article in Chinese | WPRIM | ID: wpr-351161

ABSTRACT

<p><b>OBJECTIVE</b>In order to study the effect of the polymorphism at the exon2 region of the (3-LG allele gene on milk composition and yield.</p><p><b>METHODS</b>The single-strand conformation polymorphism method (PCR-SSCP) was used to analyze for polymorphism the exon2 region of the 3-LG gene (NCBI accession number: DQ489319) in Chinese Holstein.</p><p><b>RESULTS</b>Eight SSCP patterns were detected in the fragments: ab, abc, abd, abe, abcd, abce, abde and abcde, and the patterns frequencies as follows: 0.14, 0.10, 0.27, 0.23, 0.05, 0.04, 0.11 and 0.06 (P < 0.05); Six single nucleotide polymorphism (SNP) were detected in this study: sitel C>T, site2 T>C, site3 C>T, site4 C>C, site5 C> A, site6 A>T or C, and the polymorphism infonnation content (PIC) of these SNPs were in median or high polymorphism (PIC > 0.25).</p><p><b>CONCLUSION</b>These SNPs at the exon2 region of the beta-LG gene were remarkably and affected milk performance traits (milk yield, protein and fat contents) in Chinese Holstein.</p>


Subject(s)
Alleles , Animals , Base Sequence , Cattle , Classification , Genetics , China , Exons , Female , Lactoglobulins , Genetics , Milk , Chemistry , Molecular Sequence Data , Polymerase Chain Reaction , Methods , Polymorphism, Single-Stranded Conformational
19.
Article in Chinese | WPRIM | ID: wpr-326974

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the point mutations and polymorphisms of transforming growth factor beta-induced gene (TGFBI) in Chinese patients with keratoconus and discuss the relationship between the feature of gene mutations and single nucleotide polymorphisms of TGFBI gene and keratoconus.</p><p><b>METHODS</b>Polymerase chain reaction single strand conformation polymorphism and DNA direct sequencing were performed in 30 keratoconus cases and 30 healthy controls. All 17 exons of the TGFBI gene were analyzed for point mutations and single nucleotide polymorphisms.</p><p><b>RESULTS</b>Totally two heterozygous nucleotide changes were identified in exon 12 of the TGFBI gene. The codon 535 is changed from GGA to TGA in 1 patient, leading to a substitution of glycine to a stop codon at the protein level (G535X). The codon 540 is changed from TTT to TTC in 2 patients and 1 control individual, resulting in a nonsense mutation (F54F), and is a single nucleotide polymorphism of the gene.</p><p><b>CONCLUSION</b>Mutation and polymorphisms of the TGFBI gene were detected in Chinese patients with keratoconus in this study. The results suggest that TGFBI gene might play an important role in the pathogenesis of keratoconus.</p>


Subject(s)
Adolescent , Adult , Case-Control Studies , Child , China , Extracellular Matrix Proteins , Genetics , Female , Glycine , Genetics , Humans , Keratoconus , Genetics , Male , Middle Aged , Point Mutation , Polymerase Chain Reaction , Methods , Polymorphism, Single Nucleotide , Polymorphism, Single-Stranded Conformational , Sequence Analysis, DNA , Methods , Transforming Growth Factor beta , Genetics , Young Adult
20.
Article in English | WPRIM | ID: wpr-333842

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the distribution of pathogenic C.albican genotype and Candida species in association with the severity of vulvovaginal candidiasis (VVC).</p><p><b>METHODS</b>Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) of the internal transcribed spacer analysis was employed to identify the Candida species isolated from the vaginal secretions of 198 patients with acute VVC. SSCP and GeneScan analyses of microsatellite locus I polymorphism were used to determine the genotypes of the clinical isolates of C. albican associated with VVC. All the patients were scored for clinical signs and symptoms to evaluate the severity of VVC.</p><p><b>RESULTS</b>A total of 198 Candida strains were isolated from VVC patients, including 140 (70.7%) C. albicans strains and 58 (29.3%) non-albicans strains. In the 95 patients with severe VVC and 103 with mild-moderate VVC, C.albican was detected in 62.1% and 76.6% of the patients, respectively (P=0.011). Thirty-eight microsatellite locus I genotypes were detected in 140 unrelated C. albican strains, among which the dominant genotypes 30-45 (44 strians, 31.43%) and 32-46 (23 strains, 16.43%) were the most common, followed by genotypes 30-46 (4 strains, 2.86%) and 32-47 (9 strains, 6.42%). The overall frequencies of the 4 genotypes were significantly higher in severe VVC than in mild-moderate VVC cases (77.9% vs 42.0%, P<0.001).</p><p><b>CONCLUSION</b>C. albicans remains the most common pathogenic Candia species in patients with VVC, but the non-alibcans species seem more likely to cause severe VVC. The dominant genotypes of C. albicans with a tropism for the vagina are correlated to the severity of VVC.</p>


Subject(s)
Adolescent , Adult , Candida , Classification , Candida albicans , Genetics , Candidiasis, Vulvovaginal , Microbiology , Female , Genotype , Humans , Middle Aged , Polymerase Chain Reaction , Methods , Polymorphism, Single-Stranded Conformational , Severity of Illness Index , Young Adult
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