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Sequence-specific Primers (sequence-specific + primer)
Selected AbstractsAmplified fragment length polymorphism-derived microsatellite sequence linked to the Pch1 and Ep-D1 loci in common wheatPLANT BREEDING, Issue 1 2003J. Z. Groenewald Abstract Amplified fragment length polymorphism (AFLP) markers linked to the Aegilops ventricosa -derived chromosome segment in ,VPM1' on which the eyespot resistance gene, Pch1, and the endopeptidase gene, Ep-D1b, occur were identified. One marker was isolated from the gel, cloned and sequenced. Sequence analysis revealed a microsatellite repeat motif. Sequence-specific primers were designed to amplify a product containing the repeat motif, and the microsatellite marker was tested for cosegregation with the Ep-D1b allele. Distinct alleles were produced by the Pch1 sources, normal wheat and wheat containing the Lr19 translocation. A recombination frequency of 0.02 was calculated between the microsatellite marker and Ep-D1. [source] No association of SUMO4 M55V with autoimmune diabetes in Asian-Indian patientsINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 2 2007S. K. Sedimbi Summary Autoimmune diabetes [type 1 diabetes mellitus (T1DM), latent autoimmune diabetes in adults (LADA) and part of malnutrition-related diabetes] has been shown to have genetic predisposition. Studies in IDDM 5 have lead to the discovery of a novel polymorphism 163 A,G, of SUMO4 (small ubiquitin-related modifier) gene, associated with risk to T1DM in Asians, but not in Caucasians. We studied patients with T1DM (n = 134), patients with LADA (n = 101), patients with malnutrition-modulated diabetes mellitus (n = 66) and patients with fibrocalculous pancreatic diabetes (n = 43) and healthy controls subjects (n = 114) from Cuttack, India. Polymerase chain reaction,sequence-specific primer (PCR-SSP) was used to amplify the 163 A,G sequences. Restriction fragment length polymorphism (RFLP) was performed using restriction enzyme Taq I (PCR-RFLP). Differences in the allelic frequencies of the A and the G alleles were tested statistically using Fisher's exact test or chi-squared test wherever appropriate. P -values were considered significant when equal to or less than 0.05. No significant association was detected between SUMO4 M55V and T1DM susceptibility in Asian-Indians. Comparison of the A and G alleles with HLA DR3-DR4 did not result in any significant P -values. No significant association was found between SUMO4 M55V and LADA or malnutrition-related diabetes mellitus (MRDM). Our results show that Asian-Indians with T1DM are different from other Asian populations. Asian-Indians show more similarity to Caucasians with respect to the association of SUMO4 M55V variant in T1DM. Association studies on Asian-Indian patients with LADA and MRDM showed no significant difference in the presence of the A and the G alleles when compared to healthy controls. [source] A common haplotype of the C-C chemokine receptor 2 gene and HLA-DRB1*0301 are independent genetic risk factors for Löfgren's syndromeJOURNAL OF INTERNAL MEDICINE, Issue 5 2008P. Spagnolo Abstract. Aim., Sarcoidosis is a heterogeneous disorder with a strong genetic influence. Genetic factors are also thought to influence disease severity and outcome. We sought to determine whether polymorphisms within CCR2 gene predispose to Löfgren's syndrome , a clinically and genetically distinct sarcoidosis phenotype , and, importantly, whether this association is independent of the known association with the HLA-DRB1*0301 allele. Methods., We investigated 5 CCR2 variants and HLA-DRB1*0301 by sequence-specific primer (SSP) polymerase chain reaction (PCR) in 176 Spanish (76 Löfgren's syndrome, 100 controls) and 387 Swedish subjects (126 Löfgren's syndrome, 77 non-Löfgren sarcoidosis, 184 controls). Results., One of the deduced haplotypes (CCR2 haplotype 2) was associated with Löfgren's syndrome in both Spanish (OR: 2.03, uncorrected P = 0.02; permuted P = 0.041 vs. controls) and Swedish patients (OR: 3.02, uncorrected P = 0.0007; permuted P = 0.0027 vs. non-Löfgren sarcoidosis; OR: 2.46, uncorrected P = 0.0005; permuted P = 0.0031 vs. controls). HLA-DRB1*0301 allele frequency was also increased in Spanish (OR: 3.52, P = 0.0004 vs. controls) and Swedish patients with Löfgren's syndrome (OR: 10.98, P < 0.0001 vs. non-Löfgren sarcoidosis, OR: 7.71, P < 0.0001 vs. controls). Finally, multivariate analysis revealed that the CCR2 association was independent of HLA-DRB1*0301 in both Spanish (P = 0.02 vs. controls) and Swedish cohorts (P = 0.002 vs. non-Löfgren sarcoidosis, P = 0.001 vs. controls). Conclusions., This study confirms that CCR2 haplotype 2 and HLA-DRB1*0301 are independent genetic risk factors for Löfgren's syndrome. [source] Interferon-, +874A/T and interleukin-4 intron3 VNTR gene polymorphisms in Chinese patients with idiopathic thrombocytopenic purpuraEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 3 2007Xiaoli Chen Abstract Objectives:, The polarization of Th1/Th2 towards Th1 contributes to the pathogenesis of idiopathic thrombocytopenic purpura (ITP). Cytokines may play crucial roles in the pathogenesis of ITP. The purpose of this study was to investigate whether the interferon (IFN)-, +874(A/T) and interleukin-4 (IL-4) variable number of tandem repeats (VNTR) in intron3 polymorphisms may be responsible in part for genetic susceptibility to ITP. Methods:, Genotyping of IFN-, +874A/T and IL-4 intron3 VNTR was performed in 196 patients with ITP and 128 healthy individuals by polymerase chain reaction sequence-specific primers and direct PCR respectively. Results:, There was no association between IFN-, +874A/T and IL-4 intron3 VNTR polymorphism and ITP risk when all patients, as a group, were analyzed. When the patients were subdivided into two groups: childhood ITP and adult ITP, no statistical differences were found in the genotype and allele frequencies of IFN-, +874A/T and IL-4 intron3 VNTR between the two groups and the controls. Similar results were observed between acute childhood ITP, chronic childhood ITP, acute adult ITP or chronic adult ITP and the controls. Conclusion:, These polymorphisms were distributed similarly between the patients with ITP and the controls, demonstrating that these two candidate gene polymorphisms are not attributed to ITP susceptibility. [source] The life cycles of the temperate lactococcal bacteriophage ,LC3 monitored by a quantitative PCR methodFEMS MICROBIOLOGY LETTERS, Issue 1 2000Merete Lunde Abstract We present here a new and general approach for monitoring the life cycles of temperate bacteriophages which establish lysogeny by inserting their genomes site-specifically into the bacterial host chromosome. The method is based on quantitative amplification of specific DNA sites involved in various cut-and-join events during the life cycles of the phages (i.e. the cos, attP, attB, attL and attR sites) with the use of sequence-specific primers. By comparing the amounts of these specific DNA sites at different intervals, we were able to follow the development of the lytic and lysogenic life cycles of the temperate lactococcal bacteriophage ,LC3 after infection of its bacterial host Lactococcus lactis ssp. cremoris IMN-C18. [source] Distinct MHC class I and II alleles are associated with hepatitis C viral clearance, originating from a single sourceHEPATOLOGY, Issue 1 2004Susan M. McKiernan The role of cytotoxic T lymphocyte responses, restricted by human leukocyte antigen (HLA) class I alleles, is recognized as highly significant in the successful clearance of hepatitis C virus (HCV). The frequency of class I alleles in females inoculated with HCV genotype 1b from a single source was examined for an association with outcome. Class I typing was performed using polymerase chain reaction sequence-specific primers in 227 female subjects: 141 had chronic infection and 86 had viral clearance. Statistical analysis included ,2 testing and multiple logistic regression analysis. A*03, B*27, and Cw*01 occurred more frequently in those with viral clearance (39.5%, 14%, and 9.3%, respectively) compared with those with chronic infection (19.1%, 2.1%, and 1.4%, respectively; P , .005). B*08 occurred more often in those with chronic infection compared with viral clearance (39.7% vs. 19.8%; P = .002). In combination with previously reported class II allele associations, over 75% that successfully eliminate HCV carry either A*03, DRB1*0101, or *0401, compared with only 37% of those with chronic infection (P < .0001). The haplotypes A*03-B*07-DRB1*15-DQB1*0602 and A*02-B*27-Cw*01-DRB1*0101-DQB1*0501 are associated with viral clearance (P = .004 and .01, respectively). By multiple logistic regression analysis, the alleles A*03, B*27, DRB1*0101, *0401, and *15 are associated with viral clearance, and B*27 has the strongest association (odds ratio [OR] 7.99). The haplotype A*01-B*08-Cw*07-DRB1*03011-DQB1*0201 is associated with chronic infection (P = .002), being independent for DQB1*0201 (OR 0.27). In conclusion, certain class I alleles are associated with outcome in this homogenous cohort. More significantly, either HLA-A*03, -DRB1*0101, or -*0401 are carried by an overwhelming majority of those subjects who successfully clear HCV. (HEPATOLOGY 2004;40:108,114.) [source] Lack of association between HLA-A, -B and -DRB1 alleles and the development of SARS: a cohort of 95 SARS-recovered individuals in a population of Guangdong, southern ChinaINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 1 2008P. Xiong Summary Severe acute respiratory syndrome (SARS), caused by infection with a novel coronavirus (SARS-CoV), was the first major novel infectious disease at the beginning of the 21st century, with China especially affected. SARS was characterized by high infectivity, morbidity and mortality, and the confined pattern of the disease spreading among the countries of South-East and East Asia suggested the existence of susceptible factor(s) in these populations. Studies in the populations of Hong Kong and Taiwan showed an association of human leucocyte antigen (HLA) polymorphisms with the development and/or severity of SARS, respectively. The aim of the present study was to define the genotypic patterns of HLA-A, -B and -DRB1 loci in SARS patients and a co-resident population of Guangdong province, southern China, where the first SARS case was reported. The samples comprised 95 cases of recovered SARS patients and 403 unrelated healthy controls. HLA -A, -B and -DRB1 alleles were genotyped using polymerase chain reaction with sequence-specific primers. The severity of the disease was assessed according to the history of lung infiltration, usage of assisted ventilation and occurrence of lymphocytopenia. Although the allelic frequencies of A23, A34, B60, DRB1*12 in the SARS group were slightly higher, and A33, -B58 and -B61 were lower than in the controls, no statistical significance was found when the Pc value was considered. Similarly, no association of HLA alleles with the severity of the disease was detected. Thus, variations in the major histocompatibility complex are unlikely to have contributed significantly to either the susceptibility or the severity of SARS in the population of Guangdong. [source] Single nucleotide polymorphisms of cytokine genes in the healthy Slovak populationINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 4 2007J. Javor Summary Cytokines are molecules that control and modulate the activities of numerous target cells via binding to specific receptors. The observed differences in the cytokine production among individuals can be, at least partially, explained by gene polymorphisms. Several cytokine gene polymorphisms have been identified to play a role in susceptibility to various diseases, including autoimmune, infectious, allergic or cardiovascular diseases. The aim of the current study was to determine allele and genotype frequencies of 22 polymorphisms in 13 cytokine genes in the healthy Slovak population and to compare them with data available from six populations from Central and Southern Europe. A polymerase chain reaction with sequence-specific primers was used to genotype polymorphisms within genes encoding IL-1,, IL-1,, IL-1R, IL-1RA, IL-4R,, IL-12, IFN-,, TGF-,, TNF-,, IL-2, IL-4, IL-6 and IL-10 in a sample of 140 unrelated Slovak subjects. The allelic distribution of all polymorphisms in the Slovak population was very close to that in the geographically and historically closest populations in Central Europe , the Czech and the Polish. However, several differences were found between the Slovak and four populations from Southern Europe. The obtained data represent a basis for further studies on association of cytokine gene polymorphisms with some diseases. [source] A single amino acid exchange shifts the serological reactivity of the novel HLA-B*4442 allele product from HLA-B44 to HLA-B21INTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 3 2006F. Mrazek Summary A novel HLA-B (human leukocyte antigen-B) allele, HLA-B*4442, was identified both in a Czech patient with leukaemia and in his mother. The presence of a novel allele was initially suspected because conflicting results were obtained by serological and DNA typing techniques. The HLA typing using the polymerase chain reaction,sequence-specific primers (PCR,SSP) at the two-digit level indicated an allele belonging to the HLA-B*44 group, whereas serological typing indicated HLA-B21. Typing with PCR,sequence-specific oligonucleotides (PCR,SSO) resulted in a unique reaction pattern that could not be assigned to a known allele, PCR,SSP typing at the four-digit level did not match any known B*44 allele, either. The sequencing-based typing of the HLA-B locus then revealed the novel B*4442 allele that is identical with B*4405 except a single C,G nucleotide exchange at position 572. This exchange results in an amino acid substitution from serine to tryptophan at position 167 of the expressed HLA-B protein. The B21 serological reactivity of the novel B*4442 allele product was confirmed by employing an additional serological panel of typing sera. Our findings support previous reports claiming that serine at the position 167 in the alpha-2 domain of the HLA-B protein is a major determinant of the HLA-B44(12) serological epitope. [source] Use of real-time gene-specific polymerase chain reaction to measure RNA expression of three family members of rat cytochrome P450 4AJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 3 2001Kimberly B. Bleicher Abstract Exposure of rats to peroxisome proliferators induces members of the cytochrome P450 4A (CYP4A) family. In rats, the CYP4A family consists of four related genes, CYP4A1, CYP4A2, CYP4A3, and CYP4A8. We are specifically interested in examining CYP4A1, CYP4A2, and CYP4A3, each of which is expressed in a tissue-dependent and sex-dependent manner. While CYP4A1 is sufficiently different from the other two members to enable relatively easy specific quantitation, the close similarity between CYP4A2 and CYP4A3 makes quantitative discrimination difficult. We have combined a fluorescent real-time PCR assay (TaqMan®) with the sequence-specific mismatch amplification mutation assay (MAMA) to allow us to carry out specific quantitation of all three members of this family. The assay is designed such that a single fluorescent TaqMan® probe binds to all three gene products, while specificity is conferred by sequence-specific primers. This specific MAMA technique takes advantage of the ability of Taq polymerase to distinguish between the two cDNAs based on mismatches at the 3, end of a PCR primer. In the 84-base PCR product used for this assay, there is only a single-base difference between CYP4A2 and CYP4A3. Despite this similarity, there is at least a 1000-fold discrimination between the two sequences, using CYP4A2 or CYP4A3 specific standards. Analysis of rat liver RNA from both sexes demonstrates that this discrimination is also achieved in complex RNA mixtures. This technique should be broadly applicable to other areas of research such as allelic discrimination, detecting mutational hotspots in tumors, and discrimination among closely related members of other gene families. © 2001 John Wiley & Sons, Inc. J Biochem Mol Toxicol 15:133,142, 2001 [source] HLA typing in Taiwanese patients with oral submucous fibrosisJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 4 2004Hsin-Ming Chen Background:, A significant association of certain human leukocyte antigens (HLA) and haplotypic pairs with oral submucous fibrosis (OSF) has been reported. However, controversial result of no HLA association with OSF has also been reported. In this study, the phenotype and haplotype frequencies of HLA-A, -B, -C, -DRB1, and -DQB1 in 135 Taiwanese OSF patients were calculated and compared with those in 540 healthy control Taiwanese. Methods:, The analysis of HLA-A, -B, and -C antigens, and of HLA-DRB1 and -DQB1 alleles in OSF patients and healthy control subjects, was performed by serologic typing and DNA typing using polymerase chain reaction with sequence-specific primers (PCR-SSP), respectively. Results:, We found that the phenotype frequency of HLA-B76 (3.0%) in OSF patients was significantly greater than that (0%) in healthy control subjects (corrected P (Pc) = 0.000). In addition, the haplotype frequencies of HLA-B48/Cw7 (3.0%), -B51/Cw7 (6.7%), and -B62/Cw7 (8.2%) in OSF patients were significantly greater than the corresponding haplotype frequencies (0, 0.7, and 1.9%, respectively) in healthy control subjects (Pc = 0.000). The relative risk (RR) values of haplotypes B51/Cw7 (9.57) and B62/Cw7 (4.7) were greater than the RR values of phenotypes B51 (1.81), B62 (2.31), and Cw7 (1.91) in OSF patients. In addition, the etiologic fraction (EF) value of haplotype B51/Cw7 (0.63) was higher than the EF values of phenotypes B51 (0.2) and Cw7 (0.59). Conclusions:, We conclude that some Taiwanese areca quid (AQ) chewers with particular HLA phenotypes and haplotypes are prone to have OSF. In addition, some particular HLA haplotypes may play more important roles than the individual HLA phenotypes for the genetic susceptibility to OSF. However, the significantly increased HLA phenotype B76 and three of the common HLA haplotypes detected are present in only about 20% of incident cases of OSF. [source] HLA-DRB1*08 allele may help to distinguish between type 1 diabetes mellitus and type 2 diabetes mellitus in Mexican childrenPEDIATRIC DIABETES, Issue 1 2007Ana L Rodríguez-Ventura Background:, It may be difficult to distinguish type 1 diabetes mellitus (T1DM) from type 2 diabetes mellitus (T2DM) in the pediatric population. Autoantibodies may help to differentiate both types of diabetes, but sometimes these are positive in patients with T2DM and negative in patients with T1DM. The human leukocyte antigen (HLA)-DR genotype has been associated with T1DM and with T2DM only in adults and in determined cases. Aim:, To determine the differences in HLA class II allele frequencies in Mexican children with T1DM and T2DM. Methods:, We included 72 children with T1DM, 28 children with T2DM, and 99 healthy controls. All were Mexican, and diabetes was diagnosed according to the clinical and laboratory criteria established by the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. The HLA-DRB1 typing was performed using polymerase chain reaction,sequence-specific oligonucleotide probe and polymerase chain reaction sequence-specific primers. Results:, We found an increased frequency of HLA-DRB1*08 and a decreased frequency of HLA-DRB1*04 in the group with T2DM vs. T1DM [p = 0.0001, odds ratio (OR) = 10.58, 95% confidence interval (CI) = 3,40.8 and p = 0.0006, OR = 0.24, 95% CI = 0.11,0.53, respectively]. No significant differences were found between HLA-DRB1 alleles in T2DM vs. controls. In the group with T1DM, there was a significantly increased frequency of the HLA-DR4 and HLA-DR3 alleles relative to controls (p = 0.0000001, OR = 3.59, 95% CI = 2.2,5.8 and p = 0.00009, OR = 4.66, 95% CI = 2.1,10.3, respectively). Conclusion:, There are significant differences in the HLA profile in Mexican children with T1DM and T2DM. HLA typing could play a role in the differentiation between both types of diabetes in this population. [source] HLA class II genetic association of type 1 diabetes mellitus in Czech childrenPEDIATRIC DIABETES, Issue 3 2001Ondrej Cinek Abstract:, To examine human leukocyte antigen (HLA) class II association of type 1 diabetes mellitus (DM) in Czech children, we performed a case,control study of 261 patients diagnosed before the age of 15 and 289 non-diabetic control children. Complete HLA-DQA1, DQB1 genotyping and DRB1*04 subtyping were carried out by polymerase chain reactions with sequence-specific primers. The effect of the DRB1*04 subtypes was studied in DRB1*04 alleles carried on DQB1*0302-DQA1*03 haplotypes. The risk was statistically evaluated by testing 2 × 2 tables, considering corrected p-values <,0.05 significant. The DQB1*0302 (odds ratio, OR = 9.0), DQB1*0201 (OR = 3.4) and DQA1*03 (OR = 7.5) alleles were significantly associated with diabetes risk, while the DQB1*0602 (OR = 0.02), DQB1*0301 (OR = 0.08), DQB1*0503 (OR = 0.13), DQB1*0603 (OR = 0.20), DQA1*01 (OR = 0.28) and DQA1*02 (OR = 0.26) alleles were significantly protective. Of the DQA1-DQB1 genotypes, we point out the extremely high risk of OR = 116 conferred by HLA-DQA1*05-DQB1*0201/DQA1*03-DQB1*0302. Among DRB1*04 subtypes, DRB1*0403 was significantly protective (OR = 0.05, CI 95% 0.01,0.45). Since none of the remaining DRB1*04 subtypes was associated with type 1 DM, our study may present another piece of evidence that the DRB1*0401 and DRB1*0404 alleles do not modify type 1 diabetes risk generally in European populations. [source] The DERAA HLA,DR alleles in patients with early polyarthritis: Protection against severe disease and lack of association with rheumatoid arthritis autoantibodies,ARTHRITIS & RHEUMATISM, Issue 3 2009Nathalie Carrier Objective To define the association of alleles encoding the HLA,DR rheumatoid arthritis (RA) protective epitope (DERAA) with the presence of RA-associated antibodies at study inclusion and with severe outcome in patients with early polyarthritis (EPA). Methods Consecutive EPA patients (n = 210) were evaluated early (mean of 4.8 months after diagnosis) and prospectively (for 30 months). HLA class II typing was performed by polymerase chain reaction using sequence-specific primers, and HLA,DR alleles DERAA, RA-associated shared epitope (SE), and non-SE/non-DERAA (neither SE nor DERAA) were identified. RA-associated antibodies identified were anti-Sa/citrullinated vimentin, anti,cyclic citrullinated peptide 2, and IgM rheumatoid factor. Severe disease was defined according to a preset threshold of joint destruction and/or functional limitation. Results DERAA and SE alleles were present in 62 and 110 of the 210 EPA patients, respectively. At 30 months, severe disease was present in 78 patients (37%). In contrast to SE alleles, DERAA alleles were not associated with the production of RA-associated antibodies, but were strongly protective against severe disease at 30 months (odds ratio 0.30, P < 0.001). DERAA alleles emerged as a strong, independent protective marker on multivariate analysis. The protective effect of DERAA was seen only in patients who did not already have erosions at study inclusion, was independent of the presence of antibodies, but was not associated with spontaneous remission. Conclusion In our EPA cohort, the presence of a DERAA sequence was a strong independent predictor of a better prognosis, but only in the absence of erosive disease that was already present at inclusion. Identification of DERAA alleles may help in managing the large subgroup of EPA patients who do not have erosions at baseline. [source] Angiotensinogen and plasminogen activator inhibitor-1 gene polymorphism in relation to chronic allograft dysfunction,CLINICAL TRANSPLANTATION, Issue 1 2005Kadriye Reis Abstract:, Chronic allograft dysfunction (CAD) is the most common cause of allograft failure in the long-term, and current immunologic strategies have little effect on this condition. The renin-angiotensin system (RAS) plays important roles progression of chronic renal disease. It is thought that plasminogen activator inhibitor-1 (PAI-1) functions in the RAS, in addition to involvement in thrombotic risk and fibrosis. This study investigated possible links between angiotensinogen (AGT) genotypes (M235T/MM, MT, TT) and PAI-1 genotypes (4G4G, 4G5G, 5G5G) and CAD assessments of both types of polymorphism were performed in 82 renal allograft recipients. One hundred healthy subjects were also investigated for AGT polymorphism, and 80 healthy subjects for PAI-1 polymorphism. Genotypes were determined using polymerase chain reaction (PCR) sequence-specific primers, and PCR followed by restriction fragment length polymorphism analysis. Kidney recipients with CAD had significantly lower frequencies of the MM genotype and the M allele than the recipients without CAD (p < 0.05 and <0.001). The transplant recipients with CAD also had significantly lower frequencies of the 5G5G genotype and the 5G allele than those without CAD (p < 0.001 and <0.05). Determination of AGT M235T and PAI-1 genotypes prior to transplantation may help identify patients who at risk for chronic renal transplant dysfunction. [source] | |||||||||||||