No Association of Prolactin Receptor (PRLR) Gene and litter size in Gaddi Goat Breed Reared under Migratory System in Western Himalayan Ranges of Himachal Pradesh

Gaddi is the predominant Indian goat breed also known as “White Himalayan goat”, constituting 60-65% of total goats in the state of Himachal Pradesh. The polymorphism of prolactin receptor (PRLR) gene was found to have relationship with prolificacy in goats. In present study, polymorphism of intron 2 region of PRLR gene was investigated in Gaddi goats (n = 89) using PCR-SSCP and DNA sequencing approach. PCR-SSCP assay of 176 bp amplicon of intron 2 region of PRLR gene revealed polymorphism with three types of genotypes viz., AA, AB and BB with genotypic frequencies as 0.31, 0.55 and 0.14, respectively. The allelic frequency of alleles A and B were 0.59 and 0.41, respectively in all the screened goat population. Genetic diversity analysis revealed the value of Ne, Hobs, Hexp and PIC were 1.96, 0.52, 0.49 and 0.37, respectively. The Ne and Hobs values also indicated that sufficient genetic variation exists at the studied locus. FIS estimate was observed as -0.15 indicating heterozygous excess at studied locus. DNA sequencing of amplified product revealed one nucleotide mutation (T92C) in intron 2 region of PRLR gene. The mean litter size in AA, AB and AB genotypes were 1.27±0.12, 1.41±0.09 and 1.84±0.26, respectively. No significant (P>0.05) associations of PRLR genotypes with litter size were observed. Effect of season and parity were also found to be non-significant (P>0.05) on litter size. Consequently, the study on additional data based on more number of animals in diversified flock should be carried out for future association studies.

Determination of Repeat Numbers of (CA)n in Mitochondrial D-loop using Polymerase Chain Reaction-single Strand Conformational Polymorphism (PCR-SSCP) / 체질인류학회지

Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis is a kind of sensitive mutation detection method that has been usually used in field of medical genetics. A single DNA strand with a mutation or nucleotide polymorphism has a different conformation from its wild-type counterpart, and these conformational differences result in different electrophoretic mobility. In previous study of mitochondrial microsatellite instability in 50 uterine leiomyomas, PCR-SSCP showed 4 types of band mobility at (CA)n of the mitochondrial D-loop. In type 1 and 4, positions of the lower single stand of both were same but those of upper strand were different. In sequencing analysis, repeat number of (CA)n in type 1 was 4, 5 in type 2, 6 in type 3, and 4 in type 4, respectively. Without using expensive sequencing analysis, PCR-SSCP method can be used to detect the repeat number of (CA)n in mitochondrial D-loop.

BRCA1 microsatellite instability in endometrial carcinoma and its relationship with clinical pathology / 实用医学杂志

Objective To study the occurrence of BRCA1 MSI in endometrial cancer and its relationship with clinic pathologic features; to explore the correlation between MSI and protein expression in BRCA1 gene. Methods Application of PCR-SSCP and DNA sequence analysis method was used to study D17S579 and D17S1349 in 49 sporadic endometrial cancer tissues, 20 cases with endometrial atypical hyperplasia and 28 cases with normal endometrial tissues. Results In the total samples of D17S579 and D17S1349, the three groups were significantly different: 34.69%(17/49) in the endometrial cancer group, 10%(2/20) in the endometrial atypical hyperplasia group and 7.14%(2/28) in the normal endometrium group (χ2= 11.208, P = 0.004). BRCA1 MSI positive rate related to the pathology grade and clinical stage, but no relationship was found in muscular infiltration depth, lymph node metastasis and histopathology types. In the endometrial cancer group, BRCA1 MSI positive rate and BRCA1 protein expression were in moderate correlated negatively (r = -0.779, P = 0.000). Conclusion BRCA1 MSI might play a role in the development of endometrial cancer, and low expression of BRCA1 protein. BRCA1 MSI might be associated with pathology grade and clinical stages in EC.

Trans Sialidase Genes Allow Clustering of TcI Trypanosoma cruzi Mexican Isolates.

Aims: Polymorphism of the trans sialidase (TS) family of genes is common in Trypanosoma cruzi. Our goal was to cluster Mexican TcI DTU (Discrete Typing Unit) using a set of primers specific for TS genes. Methodology: The DNA of 12 Mexican T. cruzi stocks (TcI) and reference strain were amplified using the CRP-1 primer, which anneals to the conserved 5' ends of CRP (Complement Regulatory Protein), TS, and FL-160 genes, and the CRP-2 primer, which anneals to conserved region within the GPI (Glycosil Phosphatidil Inositol) anchor sequence. Amplicons were analysed using PCR-SSCP (Single Strand Conformation Polymorphims) followed by construction of a nominal matrix data (presence/absence bands) to calculated the Jaccard and Dice similarity coefficient, and clustering with UPGMA. Results: Mexican TcI stocks produced a common pattern of amplification products and cluster in a separate group to CL-Brener strain (TcVI). The PCR-SSCP revealed that within the TcI Mexican stocks there were a complex pattern, but T. cruzi from the Yucatan peninsula clustered in special and separate group. Conclusion: The CRP-1 and CRP-2 primers were helpful for the analysis of genetic traits in T. cruzi DTU I and revealed the existence of special group in Yucatan Mexico.

Rapid detection of MDR-Mycobacterium tuberculosis using modified PCR-SSCP from clinical Specimens

Objective: To design a rapid test to detect the rifampin (RIF) and isoniazid (INH) resistant mutant based on polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique that analyzes the katG, rpoB genes.Methods:tuberculosis. To determine the susceptibility of isolates to anti TB drugs, the proportional method was used. Mutations presented within the amplified products of the katG, rpoB genes were evaluated by SSCP.Results:Using proportional method, 12 (11.6%) and 9 (8.7%) isolates were resistant respectively Biochemical test as well as IS6110 targeting PCR revealed 103 clinical samples were to INH and RIF and 9 (8.7%) isolates showed resistance to both drug (multi-drug resistant tuberculosis). Three (2.9%) multi-drug resistant tuberculosis and two INH resistant isolates were detected by the PCR-SSCP and sequencing. The sensitivity and specificity of PCR-SSCP for multi-drug resistant isolates were 33% and 100%, respectively.Conclusions:Complete agreement between SSCP and sequencing can indicate that resistance-associated mutations have occurred in other genes except our considered genes.

Rapid detection of MDR-Mycobacterium tuberculosis using modified PCR-SSCP from clinical Specimens

<p><b>OBJECTIVE</b>To design a rapid test to detect the rifampin (RIF) and isoniazid (INH) resistant mutant based on polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique that analyzes the katG, rpoB genes.</p><p><b>METHODS</b>Biochemical test as well as IS6110 targeting PCR revealed 103 clinical samples were tuberculosis. To determine the susceptibility of isolates to anti TB drugs, the proportional method was used. Mutations presented within the amplified products of the katG, rpoB genes were evaluated by SSCP.</p><p><b>RESULTS</b>Using proportional method, 12 (11.6%) and 9 (8.7%) isolates were resistant respectively to INH and RIF and 9 (8.7%) isolates showed resistance to both drug (multi-drug resistant tuberculosis). Three (2.9%) multi-drug resistant tuberculosis and two INH resistant isolates were detected by the PCR-SSCP and sequencing. The sensitivity and specificity of PCR-SSCP for multi-drug resistant isolates were 33% and 100%, respectively.</p><p><b>CONCLUSIONS</b>Complete agreement between SSCP and sequencing can indicate that resistance-associated mutations have occurred in other genes except our considered genes.</p>

Screening for Caveolin-3 gene polymorphism in Chinese Han diabetic patients / 实用医学杂志

Objective To observe the difference of caveolin-3(CAV3) gene polymorphism between normal people and diabetic patients in Chinese Han population. Methods Exon gene polymorphism in 50 normal people and 50 T2DM patients were detected by PCR-SSCP. Results The cumulative incidence rate of electrophoretic variation in T2DM patients was 48%, while cumulative incidence rate of normal people was 7%(P<0.001). It was proved that in the variant bands, there were base variant. Conclusions The variant base number of CAV3 gene in human T2DM samples are significantly more than the normal which can be preliminary detected by PCR-SSCP. It indicates that CAV3 gene polymorphism may be one of the genetic backgrounds for the occurence of Chinese T2DM.

Identification of Clematis species by PCR-SSCP analysis / 中国药学杂志

OBJECTIVE: To establish a rapid and accurate identification method to distinguish species of Clematis. METHODS: First, the registered ITS sequences of Clematis were seeked in the genbank database and compared with those of three species of Clematis' to confirm the stable specific identification site. Second, specific primers were designed, and PCR-SSCP was used to test Clematis. RESULTS: The fingerprints of the samples existed significant differences, indicating that our method was able to tell apart the different species of Clematis. CONCLUSION: PCR-SSCP has the advantages of high specificity and good reproducibility, therefore, it lays the foundation for the further development of accurate identification of Clematis.

Identificación de la mutación R579X en el exón 18 del gen RB1, en pacientes con lesiones cervicales asociadas a infección por virus del papiloma humano / Identification of the R579X mutation in exon 18 of the RB1 gene in patients with cervical infection associated with human papilloma virus

Identificar la mutación R579X ubicada en el exón 18 gen RB1, en pacientes con lesiones cervicales asociadas a infección por virus de papiloma humano mediante PCR-SSCP, PCR-RFLP y secuenciamiento. Estudio de 72 muestras de hisopado/cepillado de cuello uterino provenientes de 36 mujeres sanas, y 36 con infección por virus de papiloma humano y con presencia de lesiones cervicales a las cuales se les realizó la extracción de ADN, la amplificación del exón 18 del gen RB1 mediante PCR, y el análisis mediante PCR-SSCP, Secuenciamiento y PCR-RFLP. El análisis por PCR-SSCP reveló dos patrones diferentes de corrida y mediante el secuenciamiento se logró identificar la mutación R579X en el exón 18 del gen supresor de tumores RB1 en el 30,56 por ciento de las muestras experimentales. La mutación R579X encontrada en las muestras experimentales conlleva a la formación de una proteína truncada no funcional, y aunado a esto, el virus de papiloma humano podría estar favoreciendo a la inmortalización de las células que presentan dicha mutación

To identify the mutation R579X in exon 18 located RB1 gene in patients with cervical lesions associated with human papilloma virus infection by PCR-SSCP, PCR-RFLP and sequencing. Study of 72 samples of cervical brushing of the healthy women, infected with human papilloma virus and with cervical lesions that required DNA extraction, PCR amplification of exon 18 of the RB1 gene, PCR-SSCP, Sequencing and PCR-RFLP analysis. PCR-SSCP analysis revealed two different band patterns. We indentified the R579X mutation in exon 18 of the RB1 tumor suppressor gene in 30.56 percent of the experimental samples. The mutation found in the experimental samples leads to the formation of a non-functional truncated protein, in adition, human papilloma virus infection might contributed to the immortalization of cells with this mutation

Allelic frequency of the Kappa-Casein gene in Colombian and creole cattle breeds / Frecuencia alélica del gen Kappa-Caseína en bovinos criollos y colombianos / Freqüência do alelo de gene de Kappa-Caseína em bovinos crioulos e colombianos

Colombia has one of the most genetically diverse creole cattle populations, with eight creole breeds and two improved creole (Colombian) breeds. A high demand for meat and milk has led to the inevitable selection of highly productive cattle and the introduction of foreign breeds. Unfortunately, these breeds are often ill-suited for tropical conditions. These factors threaten the size of the creole livestock population, which is considered part of Colombia's national heritage. Objective: to estimate the allelic frequencies of the Kappa-Casein gene (CNS3) in Colombian creole cattle breeds (GCC). Methods: a total of 354 blood samples were taken from 30 animals of each of the following breeds: Blanco Orejinegro (BON), Caqueteño (CQT), Casanareño (CAS), Horned Costeño (Costeño con Cuernos, CCC), Chino Santandereano (ChS), Hartón del Valle (HV), Romosinuano (ROM), and Sanmartinero (SM), each representing the 8 established ''criollo'' (creole) breeds; the Lucerna (LUC) and Velasquez (VEL) representing the two Colombian improved breeds; and Brahman and Holstein as control breeds. DNA was extracted by a salting-out procedure and a 453 bp fragment on chromosome 6 was amplified by PCR. CSN3 alleles were identified using single strand conformation polymorphism (SSCP) and their sequence compared with those of the Genebank for Bos taurus and Bos indicus. Results: higher frequencies for allele variants of CSN3 A (0.39) and B (0.41) were found relative to the frequencies of I (0.038), G (0.095), A1 (0.025), E (0.006), and N (0.006). The allele of interest (CSN3 B) had a high frequency in the CCC (0.81), ROMO (0.66), CQT (0.55), ChS (0.48), and VEL (0.43) breeds. Conclusions: these findings suggest that Colombian creole breeds harbor a high genetic diversity which enriches its gene pool and warrants future conservation efforts to protect its integrity.

Colombia es uno de los países más diversos en recursos genéticos criollos. Posee ochos razas de ganado criollo (GCC) y dos razas de criollo mejorado o razas colombianas. La creciente demanda de alimentos ha generado una forzosa selección de individuos altamente productivos e introducción de razas foráneas (Holstein y Brahman) poco adaptadas a condiciones tropicales, lo que ha puesto en riesgo el tamaño efectivo del ganado criollo, considerado patrimonio nacional. Objetivo: estimar la frecuencia alélica del gen (CNS3) de la Kappa-Caseína en el (GCC). Métodos: se usaron 354 muestras de sangre de ocho razas bovinas criollas (30 individuos por raza): Blanco Orejinegro (BON), Caqueteño (CQT), Casanareño (CAS), Costeño con Cuernos (CCC), Chino Santandereano (ChS), Hartón del Valle (HV), Romosinuano (ROMO) y Sanmartinero (SM), dos colombianas Lucerna (LUC) y Velásquez (VEL) y dos controles Brahman y Holstein. Con el fin de estimar la frecuencia de los alelos k-caseína (k-CN) se amplificó un fragmento de 453 pb para k-CN (cromosoma 6). Los alelos se identificaron mediante la técnica PCR-SSCP. Resultados: se encontró mayor frecuencia para las variantes de k-CN A (0.39) y B (0.41), en comparación a I (0.038), G (0.095), A1 (0.025), E (0.006) y N (0.006). El alelo de interés k-CN B presentó alta frecuencia en las razas CCC (0.81), ROMO (0.66), CQT (0.55), ChS (0.48), y VEL (0.43). Conclusiones: la alta frecuencia del alelo de interés del gen k-CN ratifica al GCC como alternativa viable en esquemas sostenibles de producción de leche de mejor calidad y corrobora la necesidad de evaluación y caracterización de recursos zoogenético, como primer paso para su conservación.

A Colômbia é um dos países mais diversificado em recursos genéticos crioulos, tem oito bovinos da raça nativa e duas raças melhoradas ou raças crioulo colombiano (GCC). A alta demanda por alimentos tem levado a uma seleção forçada das pessoas altamente produtivas e/ou introdução de raças estrangeiras mal adaptados às condições tropicais, que têm prejudicado o tamanho efetivo de animais considerados património crioulo. Objetivo: estimar a frequência do alelo do gene Kappa-Caseína (CNS3) na (GCC). Métodos: foram utilizados 354 amostras de sangue de oito raças nativas (30 indivíduos por raça): Blanco Orejinegro (BON), Caqueteño (CQT), Casanareño (CAS), Costeño con Cuernos (CCC), Chino Santandereano (ChS), Hartón del Valle (HV), Romosinuano (ROM) e Sanmartinero (SM), dois Colombianas Lucerna (LUC) e Velásquez (VEL) e dois controles (Brahman and Holstein). Para estimar a freqüência de alelos de κ-caseína (CSN3), amplificaram um fragmento de 453 pb para CSN3. Os alelos foram identificados por PCR-SSCP. Resultados: encontramos uma maior freqüência de variantes do CSN3 A (0.39) e B (0.41), comparado com I (0.038), G (0.095), A1 (0.025), E (0.006) e N (0.006). O alelo de interesse CSN3 B apresentou alta freqüência em raças CCC (0.81), ROMO (0.66), CQT (0.55), CHS (0.48) e VEL (0.43). Conclusões: estes resultados sugerem que o CCG é um recurso genético, que abriga uma grande diversidade genética e apoia a necessidade de avaliação e caracterização dos recursos genéticos animais como um primeiro passo para a conservação.

Drug resistance of Mycobacterium tuberculosis strains in southern Brazil / Estudo da resistência de cepas de Mycobacterium tuberculosis aos antimicrobianos no sul do Brasil

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.

Establishment and application of multi-PCR-SSCP assay for rapid detection of isoniazid- and rifampin-resistance Mycobacterium tuberculosis isolates / 中华微生物学和免疫学杂志

Objective To establish multi-PCR-single strand conformational polymorphism analysis (mPCR-SSCP) for rapid detection of isoniazid (INH) and rifampin (RIF) resistance associated katG,inhA and rpoB genes of Mycobacterium tuberculosis isolates.Methods The INH-and RFP-resistance of 134 isolates was determined by using drug susceptibility test.The primers were designed for detecting INH and RFP resistance-associated katG,inhA and rpoB gene in the isolates by mPCR-SSCP.PCR-DS technique was applied to detect the mutations in katG,inhA and rpoB genes.All the results from different assays were subsequently analyzed as well as compared.Results All of the 134 tested isolates had katG,inhA and rpoB genes.Of the 134 isolates,42 (31.3％) and 45 (33.6％) strains were INH-and RFP-resistant,respectively.The results of mPCR-SSCP and PCR-DS showed that all the 92 INH-susceptible isolates had no mutation in katG and inhA genes with 100％ specificity.In the 89 RFP-susceptible isolates,2 and 1 had mutation in rpoB genes confirmed by mPCR-SSCP and PCR-DS with 97.8％ or 98.9％ specificity,respectively.Among the 42 INH-resistance isolates,33 and 36 strains had the mutations in katG and/or inhA genes due to the results of mPCR-SSCP and PCR-DS with 78.6％ or 85.7％ sensitivity,respectively.The results of mPCR-SSCP and PCR-DS also demonstrated that in the 45 RFP-resistance isolates,41 and 43 strains had the mutations in rpoB gene with 91.1％ or 95.6％ sensitivity,respectively.Conclusion The mPCR-SSCP established in this study can be used to rapidly detect INH and RFP-resistance associated mutations in katG,inhA and rpoB genes of M.tuberculosis with convenience,specificity and sensitivity,which shows a good prospect for application in clinic.

Kappa casein genotypes and curd yield in Holstein cows / Relación entre los genotipos de la kappa caseína de vacas holstein y el rendimiento en cuajada / Relações entre os genótipos da kappa caseína em vacas holstein a rendimento de requeijão

The objective of this study was to relate kappa casein (CSN3) genotypes with curd yield (RC) and total milk protein (PTP) in Holstein cows located in the high tropics in Narino, Colombia. Twenty seven animals were used to establish the mentioned relationships. The genotype of each animal was determined by PCR - SSCP. Variables were analyzed using a linear model which included the fix effects of genotype, lactation stage, and their interaction. Age of the cow and fat percentage in milk were used as covariates. The results for RC indicate no interaction between genotype and lactation stage. Age was not statistically significant (p>0.05), while fat percent and genotype were significant (p<0.05). The Tukey - Kramer test indicated differences between the BB genotype, compared to homozygous AA and heterozygous AB. The BB genotype resulted in the best performance, requiring the least amount of milk to produce one kg of curd. As for protein content, differences were significant (p<0.05) for the effect of genotype and lactation stage: the homozygous BB had the highest percentage of milk protein during the final (third) stage of lactation.

El presente estudio tuvo como objetivo determinar las relaciones entre los genotipos para Kappa Caseína (CSN3), el rendimiento industrial en cuajada (RC) y el porcentaje total de proteína (PTP) en vacas Holstein del Trópico Alto de Nariño-Colombia. El genotipo de cada animal fue determinado molecularmente con la técnica PCR - SSCP. Para establecer las relaciones antes indicadas se utilizaron 27 unidades experimentales. Las variables fueron analizadas mediante un modelo lineal en el que se incluyeron los efectos fijos del genotipo, el tercio de lactancia, la interacción entre estos dos factores y como covariables, la edad del animal y el porcentaje de grasa en la leche. Los resultados para RC indicaron que no existe interacción entre los genotipos y el tercio de lactancia. La edad del animal no fue estadísticamente significativa (p>0.05), mientras que la covariable porcentaje de grasa y el genotipo resultaron significativos (p<0.05). La prueba estadística de Tukey - Kramer indicó diferencias entre el genotipo BB, respecto al homocigoto AA y al heterocigoto AB, siendo el primero el de mejor rendimiento, al requerir la menor cantidad de leche para producir un kilogramo de cuajada. En cuanto al porcentaje de proteína, se encontraron diferencias estadísticamente significativas (p<0.05) únicamente por efecto del genotipo y del tercio de lactancia, siendo el homocigoto BB el que presentó mayor porcentaje de proteína en el tercer tercio de lactancia.

Este estudo teve como objetivo determinar as relações entre os genótipos para Kappa Caseína (CSN3), a rendimento industrial em requeijão (RC) e a percentagem de proteína total (PTP) em vacas Holstein do trópico alto do Nariño Colômbia. O genótipo de cada animal foi determinado molecularmente mediante a técnica PCR-SSCP. Para estabelecer as relações descritas acima, foram utilizadas 27 unidades experimentais e um modelo linear que incluiu os efeitos fixos do genótipo, o terço da lactação, a interação entre estes dois fatores, a idade do animal e o percentual de gordura no leite como covariáveis. Os resultados para (RC) indicaram ñao existe interação entre os genótipos e o terço da lactação, a idade do animal não foi estatisticamente significativa (p>0.05), mas a covariável percentagem de gordura, e o genótipo foram estatisticamente significativos (p<0.05). O teste estatístico de Tukey - Kramer indicou as diferenças entre o genótipo BB em relação a o homozigoto AA, heterozigoto AB, apresentando o primeiro o melhor desempenho porque precisou a menor quantidade de leite para produzir um quilo de requeijão. Em relação ã percentagens de proteína foram encontradas diferenças estatisticamente significativas (p<0.05) pelo efeito do genótipo e do terço de lactação, sendo o homozigoto BB o que a presenteou maior percentagem de proteína, no terceiro terço da lactação.

Distribución del polimorfismo del codón 72 del gen p53 en lesiones de cuello uterino / Distribution of codon 72 polymorphism of p53 gene in cervical lesions

Determinar la distribución del polimorfismo del codón 72 del gen p53 en pacientes que presentan lesiones cervicales asociadas a infección por VPH. Estudio descriptivo de corte transversal donde se procesaron 118 muestras del área genital femenina, de 59 mujeres sanas (controles) y 59 con lesiones cervicales NICI-NICII-NICIII y Ca in situ (casos), para la extracción y purificación del ADN. Se amplificó el exón 4 del gen p53, para la genotipificación del codón 72 mediante la técnica PCR-SSCP. Facultad de Ciencias, Laboratorio de Biología y Medicina Experimental LABIOMEX, Universidad de Los Andes. Mérida, Estado Mérida, Venezuela. La PCR-SSCP permitió determinar la frecuencia de los genotipos homocigotos arginina (Arg/Arg), prolina (Pro/Pro) y heterocigoto prolina/arginina (Pro/Arg). Para los casos el genotipo Arg/Arg tuvo una frecuencia de 32,20 por ciento y para los controles de 50,85 por ciento. El genotipo Pro/Pro se encontró en 5,09 por ciento de los casos y 11,86 por ciento para los controles. El genotipo Pro/Arg tuvo una distribución de 62,71 por ciento para los casos y 37,29 por ciento para los controles. En este estudio no se encontró una relación estadísticamente significativa entre la presencia del genotipo Arg/Arg y el desarrollo de lesiones cervicales

To determine the distribution of the polymorphism of the codon 72 of the gene p53 in patients that present cervical lesions associated to infection by VPH. Descriptive and transversal study through assessment of 118 samples of the genital feminine area were processed, of 59 healthy (control) women and 59 with cervical lesions NICI-NICII-NICIII and Ca in situ (cases), for the extraction and purification of the DNA. The exon 4 of the gene p53 was amplified, for the genotipification of the codon 72 by means of technical PCR-SSCP. Facultad de Ciencias, Laboratorio de Biologia y Medicina Experimental LABIOMEX, Universidad de Los Andes. Merida, Estado Merida, Venezuela. PCR-SSCP allowed determining the frequency of the homozygotes genotypes arginine (Arg/Arg), proline (Pro/Pro) and heterozygotes proline /arginine (Pro/Arg). For the cases the genotype Arg / Arg had a frequency of 32.20 percent and for the controls of 50.85 percent. The genotype Pro/Pro was in 5.09 percent of the cases and 11.86 percent for the controls. The genotype Pro/Arg had a distribution of 62.71 percent for the cases and 37.29 percent for the controls. In this investigation there was not a statistically significant relationship among the presence of the genotype Arg/Arg and the development of cervical lesions

Association of IGF-I gene polymorphisms with milk yield and body size in Chinese dairy goats

The association of IGF-I gene polymorphisms with certain traits in 708 individuals of two Chinese dairy-goat breeds (Guanzhong and Xinong Saanen) was investigated. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods were employed in screening for genetic variation. Two novel mutations were detected in the 5'-flanking region and in intron 4 of IGF-I gene, viz., g.1617 G > A and g.5752 G > C (accession D26119.2), respectively. The associations of the g.1617 G > A mutation with milk yield and the body size were not significant (p > 0.05). However, in the case of g.5752 G > C, Xinong Saanen dairy goats with the CG genotype presented longer bodies (p < 0.05). Chest circumference (p < 0.05) was larger in Guanzhong goats with the GG genotype. In Xinong Saanen dairy goats with the CC genotype, milk yields were significantly higher during the first and second lactations (p < 0.05). Hence, the g.5752 G > C mutation could facilitate association analysis and serve as a genetic marker for Chinese dairy-goat breeding and genetics.

PCR-SSCP Analysis of the Pronucleus Microbial Communities in Luzhou-flavor Daqu / 微生物学通报

PCR-SSCP(single-strand conformation polymorphism) was used for studying the community changes of pronucleus microorganisms in various fermenting stages of Luzhou-flavor Daqu. The results showed:(1) The pronucleus microorganism's community was similar and also had the polymorphism in each simple of various fermentation stage;(2) Different microflora had complex ecology effects of coordination and the restriction;(3) The diversity indexes of different stage of Daqu microorganisms were around 1.69～2.01,and the composition of them was stable relatively;(4) The similarity indexes were 0.67～1.00,and much higher in the approaching stages.

Rapid Selection of Halophilic Streptomonospora Strains by PCR-SSCP / 微生物学通报

To improve the efficiency of halophilic actinobacteria screening and carry out the rapid selection of targeted strains, we tested 34 strains of Streptomonospora by PCR-single strand conformation polymorphism analysis (PCR-SSCP) based on genus-specific primers for the PCR identification. This approach employs PCR with two pairs of primers located in the 16S rRNA sequence flanking two variable region, then build clustering tree according as SSCP data. Synchronously, we sequenced all the 16S rRNA partial sequences for these strains to verify them. The results showed that the PCR-SSCP analysis was an efficient, easy-to-handle and economic method for rapid selection of halophilic actinobacteria resources.

p53 Mutation and p53 Protein Expression in Gastric Cancer Tissues

PURPOSE: Variable changes occur in the progression from normal gastric epithelium to cancer, including many tumor, tumor suppressor and DNA repair genes, as well as growth factor and its receptors. The mutation and protein expression of the p53 gene may be useful prognostic factors, but their significance is still uncertain. METHODS: Specimens from 296 gastric cancer patients, treated by a curative gastrectomy, between March 1999 and April 2001, at Kyungpook National University Hospital, were used. The p53 gene mutation was assessed using a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, and the overexpression of tumor p53 protein using immunohistochemistry. The correlation between the results and clinicopathological parameters were then analyzed. RESULTS: The mutation and protein overexpression of the p53 gene were shown in 61 (20.6%) and 124 (41.9%) tumors, respectively. Of the 61 cases with a p53 mutation, 43 (70.5%) showed overexpression of the p53 protein, and of the 235 without mutation of the p53 gene, 81 (34.5%) had no overexpression of the p53 protein, and also showed statistical significance (P< 0.001). The mutation and protein overexpression of the p53 gene showed no significant differences according to age, gender, stage, location and gross type, but of the 138 intestinal and 128 of the diffuse types, 33 (23.9%) and 18 (14.1%) cases, respectively, showed p53 mutation (P=0.027); whereas, of the 150 well differentiated and 142 poorly differentiated tumors, 75 (50%) and 18 (33.8%), respectively, showed overexpression of the p53 protein. Also, of the 138 intestinal and 128 diffuse types, 71 (51.4%) and 43 (33.6%) showed overexpression of the p53 protein. There were no significant differences in the 5 year survival according to the mutation and protein overexpression of the p53 gene. CONCLUSION: The mutation and protein overexpression of the p53 gene, as assessed by PCR-SSCP and immunohistochemistry, respectively, showed a statistically significant correlation, but had little value as prognostic factors following a curative gastrectomy.

VARIATION ANALYSIS OF HPV16 CELL-TYPE-SPECIFIC ENHANCER IN CERVICAL CARCINOMA / 药物分析学报

Objective To investigate the cell-type-specific enhancer (CTSE) in HPV16 and its variation in cervical carcinoma. Methods CTSEs were detected by polymerase chain reaction (PCR) in 58 cervical carcinoma from Shaanxi province; in addition variation of CTSEs was analyzed through single-strand conformation polymorphisms (SSCP). Results HPV16 CTSEs were detectable in 34 of 58 (57%) specimens and mutant rate was 41%(14/34) and the main mutations of chosen randomly variant CTSE (CTSEv) happened at YY1 binding sites in addition to glucocoticoid response elements (GRE). Conclusion CTSE in some specimens of Shaanxi province was obviously different from that in HPV16 wild type and variant CTSE might affect the transcriptional regulation of LCR on viral P97, which regulates over-expression of viral oncogenes in cervical carcinoma.

Detection of rpsL drug-resistant gene in L-form of Mycobacterium tuberculosis by PCR-SSCP / 临床检验杂志

Objective To study the relationship between mutation of rpsL drug-resistant gene in L-forms of Mycobacterium tuberculosis and drug-resistance to streptomycin in pneumoconiosis patients.Methods A total of 52 clinical isolated strains of Mycobacterium tuberculosis L-forms were collected from 97 pneumoconiosis patients.The mutation of rpsL gene was detected by PCR-SSCP,and the drug-resistance to streptomycin was performed by routine antimicrobial susceptibility test(AST).Results The results of drug susceptibility test showed that 26 in the 52 clinical isolated strains were drug-resistant to streptomycin.The streptomycin-resistant rate was 50.00%(26/52).The gene mutation rate of rpsL detected by PCR-SSCP was 40.38%(21/52).The coincidence rate of two experimental results was 80.77%(21/26).Conclusion High detectable rate of streptomycin-resistant strains in Mycobacterium tuberculosis L-forms was found by PCR-SSCP.The application of PCR-SSCP may possess important value for guiding clinical medication of pneumoconiosis patients complicated with tuberculosis among coal workers