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Diagnóstico de linfomas cutáneos mediante detección de clonalidad por PCR-heterodúplex
Amor Vigil,Ana M.; Matos Borges,Rafael; Martínez Antu?a,Gisela;
Revista Cubana de Hematolog?-a, Inmunolog?-a y Hemoterapia , 2008,
Abstract: the clonicity detection in the lymphoproliferative syndromes by studying the rearrangement of the immunoglobulin genes and of the t-receptor cells is used to make clear if a proliferation or infiltrate of lymphocytes is malignant or not. this type of study is particularly useful in the presence of cutaneous lesions whose lymphoid or dermatological origin is difficult to define. by the pcr-heteroduplex technique, the genes of the immunoglobulin heavy chain and of the t-cell receptor chain were studied in 10 patients that presented dermatological manifestations attributable to some kind of cutaneous lymphoma. clonal rearrangement was observed in 7 patients, which allowed to confirm the diagnosis of mycosis fungoides and other types of cutaneous lymphomas. it was not possible to confirm a lymphoid process of malignant character by this technique in 3 patients who did not show clonal rearrangement. the usefulness of the study was proved when in the presence of a skin affection, it was difficult to differentiate a dermatological process from a proliferative syndrome with cutaneous manifestations.
Optimization of heteroduplex analysis for the detection of BRCA mutations and SNPs  [cached]
Lucian Negura,Dragos Peptanariu,Anca Negura,Eugen Carasievici
Analele ?tiin?ifice Ale Universit??ii Alexandru Ioan Cuza din Ia?i,Sectiunea II A : Genetica si Biologie Moleculara , 2011,
Abstract: BRCA1 and BRCA2 are tumour suppressor genes whose mutant phenotypes predispose to breast and ovarian cancer. Screening for mutations in these genes is now standard practice for hereditary breast and ovarian cancer (HBOC) cases in Europe, and permits medical follow-up and genetic counselling adapted to the needs of individuals in such families. Currently, most laboratories performing diagnostic analysis of the BRCA genes use PCR of exons and intron-exon boundaries coupled to a pre-screening step to identify anomalous amplicons. The techniques employed for the detection of mutations and SNPs have evolved over time and vary in sensitivity, specificity and cost-effectiveness. As a variant for pre-screening techniques, we chose the recently developed Surveyor heteroduplex cleavage method as a sensitive and specific technique to reveal anomalous amplicons of the BRCA genes, using only basic laboratory equipment and agarose gel electrophoresis. Here we present the detection of either mutations or SNPs within the BRCA1 exon 7, using heteroduplex analysis (HA) by mismatch-specific endonuclease, confirmed by dideoxy sequencing.
Construction of heteroduplex DNA andin vitro model for functional analysis of mismatch repair
Yi Wang,Alan Clark,Jiaxun Wang,Menghong Sun,Daren Shi
Chinese Science Bulletin , 2004, DOI: 10.1007/BF02901740
Abstract: Functional deficiency of mismatch repair (MMR) system is one of the mechanisms of tumorigenesis. With the development of the investigation and the requirement from the clinical diagnosis and treatment it is necessary to build up a method to evaluate the functional status of the whole MMR system in the concerned tumors. The original ssDNA and dsDNA from wild type (wt) bacteriophage M13mp2 and its three derivates with mutation points in thelacZα gene have been used to construct two kinds of heteroduplex DNA molecules. One named del(2) has two bases deleted in the negative strand, the other has a G·G mismatch base pair in the negative strand too. Introducing this heteroduplex DNA intoE. coli NR9162 (mutS ) without the MMR ability on the indicator plate with x-gal and IPTG, there are three kinds of plaques, mixture plaque as the characteristic phenotype of heteroduplex DNA, blue and clear plaques. If the cell extract is mismatch repair competent the percentage of the mixture plaque will decrease after incubation with these heteroduplex DNA, the repair efficiency is expressed in percentage as 100× (1 minus the ratio of percentages of mixture plaque obtained from the extract-treated sample and untreated samples), which can imply the functional status of MMR system of certain samples. After large T-antigen-dependent SV-40 DNA replication assay cell extract from TK6, a human lymphoblastoid B-cell lymphoma cell line with MMR ability, and Lovo, a human colonic carcinoma cell line with MMR deficiency have incubated with these heteroduplex DNA. The repair efficiency of TK6 to del(2) is more than 60%, to G·G is more than 50%. The Lovo efficiency to del(2) is less than 10%, to G·G is less than 20%. Therefore, in thisin vitro model used for functional analysis of mismatch repair of heteroduplex DNA as the repair target, TK6 can serve as the control for MMR proficiency and Lovo as the control for MMR deficiency. Using this model the tumor tissue from a case of hereditary nonpolyposis colorectal cancer (microsatellite instability high, MSI-H) was measured and lack of MMR ability was shown. And a case of sporadic rectal cancer (SRC) (microsatellite stability, MSS) maintains MMR proficiency. The results indicate that the model is sensitive and dependable. It could be used to measure the function status of MMR system in tumor cell and/or tissues. This is a reliable method to investigate the mechanic of tumorigenesis. It is meaningful in the observation of the role of MMR in the initiation and progression of concerned tumors.
Construction of heteroduplex DNA and in vitro model for functional analysis of mismatch repair

WANG Yi,Clark Alan,WANG Jiaxun,SUN Menghong,SHI Daren,

科学通报(英文版) , 2004,
Abstract: Functional deficiency of mismatch repair (MMR) system is one of the mechanisms of tumorigenesis. With the development of the investigation and the requirement from the clinical diagnosis and treatment it is necessary to build up a method to evaluate the functional status of the whole MMR system in the concerned tumors. The original ssDNA and dsDNA from wild type (wt) bacteriophage M13mp2 and its three derivates with mutation points in thelacZα gene have been used to construct two kinds of heteroduplex DNA molecules. One named del(2) has two bases deleted in the negative strand, the other has a G·G mismatch base pair in the negative strand too. Introducing this heteroduplex DNA intoE. coli NR9162 (mutS ) without the MMR ability on the indicator plate with x-gal and IPTG, there are three kinds of plaques, mixture plaque as the characteristic phenotype of heteroduplex DNA, blue and clear plaques. If the cell extract is mismatch repair competent the percentage of the mixture plaque will decrease after incubation with these heteroduplex DNA, the repair efficiency is expressed in percentage as 100× (1 minus the ratio of percentages of mixture plaque obtained from the extract-treated sample and untreated samples), which can imply the functional status of MMR system of certain samples. After large T-antigen-dependent SV-40 DNA replication assay cell extract from TK6, a human lymphoblastoid B-cell lymphoma cell line with MMR ability, and Lovo, a human colonic carcinoma cell line with MMR deficiency have incubated with these heteroduplex DNA. The repair efficiency of TK6 to del(2) is more than 60%, to G·G is more than 50%. The Lovo efficiency to del(2) is less than 10%, to G·G is less than 20%. Therefore, in thisin vitro model used for functional analysis of mismatch repair of heteroduplex DNA as the repair target, TK6 can serve as the control for MMR proficiency and Lovo as the control for MMR deficiency. Using this model the tumor tissue from a case of hereditary nonpolyposis colorectal cancer (microsatellite instability high, MSI-H) was measured and lack of MMR ability was shown. And a case of sporadic rectal cancer (SRC) (microsatellite stability, MSS) maintains MMR proficiency. The results indicate that the model is sensitive and dependable. It could be used to measure the function status of MMR system in tumor cell and/or tissues. This is a reliable method to investigate the mechanic of tumorigenesis. It is meaningful in the observation of the role of MMR in the initiation and progression of concerned tumors.
Divergent strains of human immunodeficiency virus type 1 circulating in India, subtyped by heteroduplex mobility assay  [cached]
Sahni A,Kapila K,Gupta R
Indian Journal of Pathology and Microbiology , 2008,
Abstract: Genomic variations in HIV-1 represent a major problem in understanding disease progression, studying drug resistance and developing effective vaccines. Heteroduplex Mobility Assay (HMA) was used for analyzing HIV-1 subtypes resulting from genetic similarity or divergence of C2 -V3 -V5 region of envelope gene between HIV-1 strains obtained from clinical samples in a tertiary care center at Pune. DNA from the PBMCs of infected individuals was amplified by nested PCR. Heteroduplexes were then formed by denaturing DNA from the unknowns with DNA from the reference strains. The results were analyzed by polyacrylamide gel electrophoresis. Out of 177 samples analyzed, 170 were of subtype C (96%). Four samples were found to be of subtype B (2.2%); in three samples, no definitive assignment of subtype was possible by HMA and these perhaps could be circulating recombinant forms (CRFs) of HIV-1. These findings may have significant implications toward development of a candidate vaccine for India.
Physical Analyses of E. coli Heteroduplex Recombination Products In Vivo: On the Prevalence of 5′ and 3′ Patches  [PDF]
Laura M. Gumbiner-Russo, Susan M. Rosenberg
PLOS ONE , 2007, DOI: 10.1371/journal.pone.0001242
Abstract: Background Homologous recombination in Escherichia coli creates patches (non-crossovers) or splices (half crossovers), each of which may have associated heteroduplex DNA. Heteroduplex patches have recombinant DNA in one strand of the duplex, with parental flanking markers. Which DNA strand is exchanged in heteroduplex patches reflects the molecular mechanism of recombination. Several models for the mechanism of E. coli RecBCD-mediated recombinational double-strand-end (DSE) repair specify that only the 3′-ending strand invades the homologous DNA, forming heteroduplex in that strand. There is, however, in vivo evidence that patches are found in both strands. Methodology/Principle Findings This paper re-examines heteroduplex-patch-strand polarity using phage λ and the λdv plasmid as DNA substrates recombined via the E. coli RecBCD system in vivo. These DNAs are mutant for λ recombination functions, including orf and rap, which were functional in previous studies. Heteroduplexes are isolated, separated on polyacrylamide gels, and quantified using Southern blots for heteroduplex analysis. This method reveals that heteroduplexes are still found in either 5′ or 3′ DNA strands in approximately equal amounts, even in the absence of orf and rap. Also observed is an independence of the RuvC Holliday-junction endonuclease on patch formation, and a slight but statistically significant alteration of patch polarity by recD mutation. Conclusions/Significance These results indicate that orf and rap did not contribute to the presence of patches, and imply that patches occurring in both DNA strands reflects the molecular mechanism of recombination in E. coli. Most importantly, the lack of a requirement for RuvC implies that endonucleolytic resolution of Holliday junctions is not necessary for heteroduplex-patch formation, contrary to predictions of all of the major previous models. This implies that patches are not an alternative resolution of the same intermediate that produces splices, and do not bear on models for splice formation. We consider two mechanisms that use DNA replication instead of endonucleolytic resolution for formation of heteroduplex patches in either DNA strand: synthesis-dependent-strand annealing and a strand-assimilation mechanism.
Genetic variability among isolates of Coconut lethal yellowing phytoplasmas determined by Heteroduplex Mobility Assay (HMA)
Marinho, Vera Lucia A.;Fabre, Sandrine;Dollet, Michel;
Tropical Plant Pathology , 2008, DOI: 10.1590/S1982-56762008000500006
Abstract: heteroduplex mobility assay (hma) was used to determine genomic diversity among african isolates of coconut lethal yellowing phytoplasmas causing cape st. paul wilt disease (cspd, ghana), lethal disease (ld, tanzania), and lethal yellowing (lym, mozambique). they were also compared to the caribbean phytoplasma associated with coconut lethal yellowing (ly). a dna fragment of 1850 bp covering the 16s rrna gene and 16/23s intergenic spacer region of each isolate was amplified with primers p1 and p7 and subsequently submitted to hma analysis for sequence variation. a pcr product amplified from gh5d (cspd isolate) as a reference was combined with each pcr product and electrophoresed on polyacrylamide gels. three groups of phytoplasmas associated with various coconut lethal yellowing diseases were identified by hma. the samples from mozambique (lym) and ghana (cspd) formed one group, which was different from the second group, ld from tanzania. these two groups were different from the third group of caribbean isolates. this grouping was consistent with the genetic diversity described in the coconut yellowing-associated phytoplasmas detected after cloning, sequencing, and phylogenetic analyses. the hma technique described here has the potential to provide a simple and rapid means to identify and to establish the diversity of isolates within the coconut lethal yellowing disease group.
Estudio molecular mediante reacción en cadena de la polimerasa y análisis de heteroduplex para el gen de la resistencia a la Rifampicina en Micobacterium tuberculosis  [cached]
Salcedo P.,Villegas S.,Vélez S.,Arango AI.
Acta Biológica Colombiana , 2001,
Abstract: El Micobacterium tuberculosis, es un bacilo intracelular gramnegativo, facultativo, no esporulado, aerobio estricto, de crecimiento lento y que requiere medio de cultivo complejo para su aislamiento y caracterización. La tuberculosis es una infección que cursa con diversas manifestaciones clínicas teniendo como órgano blanco el pulmón y amplia distribució mundial. El bacilo muestra resistencia o multiresistencia a los tratamientos convencionales. El objetivo del presente trabajo, mediante PCR y el análisis de Heteroduplex de una región del gen rpoB de M. tuberculosis, es detectar las cepas resistentes a la rifampicina.
Tejido linfoide y linfomas gástricos Lymphoid tissue and gastric lymphomas  [cached]
Rocío del Pilar López P,Rafael Enrique Andrade P
Revista Colombiana de Gastroenterologia , 2010,
Abstract: En este artículo hemos realizado una amplia revisión de los linfomas primarios gástricos, su clasificación y aspectos clínico-patológicos más importantes, haciendo énfasis especial en los linfomas MALT o asociados a las mucosas y su relación con la infección por Helicobacter pylori. In this review we describe various aspects of the primary gastric lymphomas, the classification, and the most important clinico-pathological aspects, with emphasis in mucosa associated lymphoma (MALT) and the Helicobacter pylori infection.
Manifestaciones pulmonares en pacientes con linfomas
Jesús Diego de la Campa,José Carnot Uría,Jorge Mu?ío Perurena,Raúl de Castro Arenas
Revista Cubana de Medicina , 2002,
Abstract: Se realizó un estudio descriptivo en 88 pacientes con linfomas (25 con enfermedad de Hodgkin y 63 con linfoma no Hodgkin) atendidos en el Hospital Clinicoquirúrgico "Hermanos Ameijeiras" desde abril de 1997 hasta diciembre de 1998, para identificar las manifestaciones pulmonares. Se estudiaron los síntomas y signos del aparato respiratorio y los resultados de la radiografía del tórax en todos los pacientes. En 79 de ellos se realizaron pruebas funcionales respiratorias. Predominaron los pacientes asintomáticos (69/88; 78,4 %) (p < 0,01), el síntoma respiratorio más frecuente fue la tos (6/25; 24 % en la enfermedad de Hodgkin y 10/63; 15,8 % en los linfomas no Hodgkin). Una proporción elevada de pacientes no tuvieron alteraciones al realizar examen físico respiratorio (73/88; 82,9 %) (p < 0,01), la disminución del murmullo vesicular fue la alteración más frecuente (15/88; 17 %). Se constató radiología del tórax normal en más de la mitad de los casos (53/88; 60,2 %) (p=0,025) y las adenopatías mediastinales fueron las alteraciones de mayor frecuencia (30/88; 34 %). Las alteraciones radiográficas fueron más frecuentes en la enfermedad de Hodgkin (18/25; 72 %) que en los linfomas no Hodgkin (17/63, 27 %). En las pruebas funcionales respiratorias, el 74,7 % fue normal (59/79; p < 0,01) mientras que el trastorno ventilatorio restrictivo ligero tuvo mayor presentación (9/79, 11,4 %). Los datos obtenidos evidencian que en el grupo de enfermos estudiados la frecuencia de las manifestaciones pulmonares es similar a la reportada por otros autores y que no existen síntomas ni signos respiratorios patognomónicos para cada tipo de linfoma. A descriptive study was conducted in 88 patients with lymphomas (25 Hodgkin’s disease and 63 non-Hodgkin’s lymphoma) seen at "Hermanos Ameijeiras" Clinical and Surgical Hospital from April 1997 to December 1998. Its objective was to identify the pulmonary manifestations. Symptoms and signs of the respiratory system and chest Rx results were analyzed in all the patients. Seventy-nine of them underwent respiratory function testing. Asymptomatic patients prevailed (69/88 for 78,4 %) (p < 0,01); the most frequent respiratory symptom was cough (6/25 for 24 % in Hodgkin’s disease and 10/63 for 15,8 % in non-Hodgkin’s lymphomas). A high number of patients did not present alterations when they were applied a respiratory physical exam (73/88 for 82,9 %) (p < 0,01); the reduction of vesicular breath sounds was the most frequent disturbance (15/88 for 17 %). It was observed that chest radiology was normal in more than half of the cases (53/88
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