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Mycobacterium tuberculosis (M. tuberculosis) genotyping provides valuable information related to the origin and the evolution of the isolates. This study aimed to evaluate the applicability of single-nucleotide polymorphisms (SNPs) technique for lineages identification of M. tuberculosis and compare it with mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) method. The lineages of 162 clinically isolates were evaluated using six pair primers by Multiplex-PCR based on SNPs. Among 162 isolates, 70 (43.2%) isolates were lineage 4, following that 62 (38.3%) and 22 (13.6%) isolates were lineage 3 and 2, respectively. The method could not type 8 (4.9%) isolates. Moreover, we could identify 71 out of 79 unknown isolates resulted from the MIRU-VNTR method. The results showed that the SNP typing method has the potential to determine the lineages of M. tuberculosis as a rapid laboratory screening test.

Genotype profiling has played a major role in forensics for decades. The technology for detection and discrimination has advanced substantially, from serology to DNA sequence analysis. Currently, there may be situations where there is a need for re-analysis of forensic DNA data that was produced using methodology that is no longer available. An example of this is the allele-specific oligonucleotide hybridization assays used in the 1990s. In the study presented herein, we have developed a multiplex system combining PCR and massively parallel sequencing (MPS) technologies to identify DNA polymorphisms. Our results are consistent with those found in the widely utilized AmpliType PM + DQA1 Amplification and Typing Kit originally marketed by Perkin Elmer. During the course of our studies, it became apparent that paralogous genes for two of the loci, GYPA and HBG2 (formerly HBGG), could have confounded the interpretation of the original assays, and we describe the technical solutions we developed to overcome ambiguity in genotype assignment. This study results in a novel resource enabling the re-analysis of DNA profiling results produced decades past using current day technology.

Huntington's disease (HD) is an incurable, autosomal dominant neurodegenerative disease caused by an abnormally long polyglutamine tract in the huntingtin protein. Because this mutation causes disease via gain-of-function, lowering huntingtin levels represents a rational therapeutic strategy.We searched MEDLINE, CENTRAL, and other trial databases, and relevant company and HD funding websites for press releases until April 2020 to review strategies for huntingtin lowering, including autophagy and PROTACs, which have been studied in preclinical models. We focussed our analyses on oligonucleotide (ASOs) and miRNA approaches, which have entered or are about to enter clinical trials.ASO and mRNA approaches for lowering mutant huntingtin protein production and strategies for increasing mutant huntingtin clearance are attractive because they target the cause of disease. However, questions concerning the optimal mode of delivery and associated safety issues remain. It is unclear if the human CNS coverage with intrathecal or intraparenchymal delivery will be sufficient for efficacy. The extent that one must lower mutant huntingtin levels for it to be therapeutic is uncertain and the extent to which CNS lowering of wild-type huntingtin is safe is unclear. Polypharmacy may be an effective approach for ameliorating signs and symptoms and for preventing/delaying onset and progression.

Background: Cytokines are the key regulator molecules that modulate immune response. Tumor necrosis factor (TNF- α-308 G/A and TNF-β +252 A/G ) are inflammatory cytokine that control the progression of several types of cancer. They play a vital role in both tumor progression and destruction based on their concentrations. The role of TNF-α-308 G/A and TNF-β +252 A/G gene polymorphism in the etiology of breast cancer (BC) is not clearly understood. Therefore, present study investigates the association of TNF-α -308 G/A and TNF-β +252 A/G and the clinical features with Breast cancer patients. Methods: In a case- control study, we have investigated 150 breast cancer patients and 300 age and ethnically matched healthy controls for duration of 3 years from North India. Promoter polymorphisms of tumor necrosis factor gene (TNF-α -308 G/A and TNF-β +252 A/G) were genotyped using allele specific oligonucleotide polymerase chain reaction ASO and restriction fragment length polymorphism (PCR-RFLP). The associations were evaluated by calculating the pooled odds ratio (OR) with 95% confidence interval (95% CI) using SPSS. Results: Patients with different clinico-pathological variables and healthy controls were analyzed. Significant association was observed in A allele of TNF-α -308 G/A in breast cancer patients as compared to healthy controls (p

Introduction Despite the impressive responses achieved with tyrosine kinase inhibitor (TKI) therapy, treatment resistance develops in 16-33% of patients of chronic myelogenous leukemia (CML). Of the BCR-ABL1 dependent mechanisms, mutations in the tyrosine kinase domain (TKD) are the commonest cause of resistance. Material and methods Allele specific oligonucleotide - polymerase chain reaction (ASO-PCR) was done for testing the six common TKD mutations, T315I, G250E, E255K, M244V, M351T, and Y253F. Results and conclusion TKD mutation study was done on 83 patients. Of these 44 (53%) were positive for one or more mutations. On analyzing specific mutations, E255K was the commonest mutation seen in 24 (29%) cases, followed by T315I in 23(28%) cases. Y253F mutation was not seen in the present study sample. In the present cohort of 83 patients, 29 (35%) cases were positive for single mutation, 12 (14%) had two mutations and 3 (4%) had three mutations.

Alterations of the P53 gene and human papillomavirus (HPV) infection are associated with development of oral squamous cell carcinoma (OSCC). We aimed to identify mutation of P53 exon 8 codon 282 in OSCC and correlate these with HPV infection as well as histopathological grade of OSCC. Samples of known HPV infection status were studied including oral lesion cells, formalin-fixed paraffin embedded (FFPE) tissues from OSCC and exfoliated oral cells of matched age-sex controls. P53 exon 8 mutation was detected using the polymerase chain reaction (PCR). Mutation of codon 282 was identified by allele-specific oligonucleotide typing (ASO) using EvaGreen real-time PCR. The PCR products were analyzed by gel electrophoresis and melting curve analysis. Mutation of P53 exon 8 was seen in 81.7% and 69.6% of FFPE OSCC tissues and oral lesion cells, respectively. This was significantly higher than in controls (16.7%). Frequency of mutation did not differ between HPV-positive samples (62.5% and 81.8% in oral lesion cells and FFPE tissue samples, respectively) and HPV-negative samples (73.3% and 81.5% in oral lesion cells and FFPE tissue samples, respectively). This finding is similar to P53 codon 282 mutation that was found only in FFPE tissues (35.0%) and oral lesion cells (32.6%) from both HPV-positive and negative OSCC. Interestingly, frequency of mutation was higher in well-differentiated OSCC with HPV-infection (28.1%) than without HPV (14.8%). This result demonstrated a mutation hot spot in P53 associated with oral carcinogenesis and might be useful to guide chemotherapeutic modality for HPV-associated OSCC in northeast Thailand.

Background: The picture of Vietnamese patients with essential thrombocythemia (ET) remains mostly undetermined. Our study intended to determine the frequency of JAK2V617F, CALR exon 9, and MPL exon 10 mutations as well as to analyze clinical characteristics associated with different mutational status in Vietnamese ET patients. Methods: We explored mutations of JAK2V617F, MPL, and CALR from 395 patients using allele specific oligonucleotide – polymerase chain reaction and Sanger sequencing techniques; then, the clinical and hematological features were compared according to mutation patterns. Results: We found that JAK2V617F, CALR exon 9, and MPL exon 10 mutations were present in 56.2%, 27.6%, and 1% of the 395 patients with ET, respectively. Twelve different types of CALR mutation were detected in 109 patients, with the CALR type 1 mutation (c.1099_1150del; L367fs*46) was the most common, followed by CALR type 2 mutation (c.1154_1155insTTGTC; K385fs*47). The JAK2V617F-positive patients had older age, higher white blood cell counts and higher hemoglobin levels but lower platelet counts than patients with CALR mutations or patients negative for triple tests. There was no significant difference regarding sex ratio, white blood cell counts, platelet counts and hemoglobin levels among CALR mutation subtypes. Conclusion: we reported high frequency of JAK2V617F, CALR, and MPL mutations in Vietnamese patients with ET and underscored the importance of combined genetic tests for diagnosis and classification of ET into different subtypes.

With contemporary therapeutic strategies in multiple myeloma, heretofore unseen depth and rate of responses are being achieved. These strategies have paralleled improvements in outcome of multiple myeloma patients. The integration of the next generation of proteasome inhibitors and antibody therapeutics promise continued improvements in therapy with the expectation of consistent depth of response not quantifiable by current clinical methods. As such, there is a growing need to develop adequate tools to evaluate deeper disease response after therapy and to refine the response criteria including the minimal residual disease. Several emerging techniques are being evaluated for these purposes including multi-parameter flow cytometry, allele-specific oligonucleotide polymerase chain reaction, next-generation sequencing, and imaging modalities. In this review, we highlight the recent developments and evaluate advantages and limitations of the current technologies to assess minimal residual disease. We also discuss future applications of these methodologies in potentially guiding multiple myeloma treatment decisions.

Background & objectives: Mutation of nucleophosmin (NPM1) gene in the absence of FLT3-ITD (FMS related tyrosine kinase 3 - internal tandem duplications) mutation carries a good prognosis in cytogenetically normal acute myeloid leukaemia (AML). NPM1, a multifunctional nucleolar phosphoprotein that shuttles between nucleus and cytoplasm, gets trapped in the cytoplasm when mutated. Immunohistochemical (IHC) demonstration of its aberrant cytoplasmic location (NPMc+) has been suggested as a simple substitute for the standard screening molecular method. This study was aimed to assess the diagnostic utility of IHC on formalin fixed bone marrow biopsies in comparison with the reference molecular method (allele specific oligonucleotide - polymerase chain reaction; ASO-PCR) to predict NPM1 mutation status in AML patients. Methods: NPM protein IHC was performed using mouse anti-NPM monoclonal antibody on 35 paraffin-embedded bone marrow biopsies of patients with primary AML of any French-American-British (FAB) subtype. Results of IHC were compared with those of ASO-PCR. Results: Of the 35 AML patients, 21 (60%) were positive for NPM1 exon 12 gene mutation by ASO-PCR, 19 (90.47%) of these 21 were NPMc+. Thirteen of the 35 patients were negative by both the methods. One NPMc+ patient was not detected by ASO-PCR. IHC had a sensitivity and specificity of 90 and 93 per cent, respectively, compared to the molecular screening gold standard. Interpretation & conclusions: Mutation of NPM1 determined by the widely available and inexpensive IHC agrees closely with results of the standard molecular methods. Thus, technically and financially not well endowed laboratories can provide the prognostically and potentially therapeutically important information on NPM1 mutation using IHC.

Objectives This study includes the investigation of antifungal activity of the local propolis against dermatophytes and yeast. Methods A total of 92 tissue samples were taken from patients infected with H. pylori and 102 from uninfected individuals has been included. Genetic method used for the detection of H. pylori infection by polymerase chain reaction (PCR) for glM gene identification. IL-8- 251 A/T polymorphism was detected by allele-specific oligonucleotide polymerase chain reaction (ASO- PCR). Results Among the studied samples, the results improve that genetic polymorphism of IL-8-251 A/T genotype was found in a higher frequency with confidence interval 95% in duodenal ulcer H. pylori infected patient than control. Conclusion IL-8-251 A allele notice that it has been associated with severe inflammation lead to increase severity of disease.

Chronic myeloid leukemia (CML) is amyeloprolirative disorder characterized by the Philadelphia chromosome originates from the translocation of chromosome 9 and 22 t (9;22) (q34;q11), creating a BCR-ABL fusion gene which results in the constitutive activation BCR-ABL tyrosine kinase. Patients of BCR-ABL fusion gene positive in (CML) are treated by an effective therapy called Imatinib (Gleevec). Point mutations alter the conformation of the ATP binding site that disturbs the binding of therapy to its target which leading to imatinib resistance. Fifty CML patients were collected from Nanakali Hospital in Erbil city which were diagnosed by physicians. The age of the patients from 9 to 80 years, 62% were male and 39% were female, median age of the patients were 38 years. The present study deals with two different aspects; conventional cytogenetic (G-banding) analysis to diagnose the Philadelphia chromosome as a (CML) marker and using Allele Specific Oligonucleotide Polymerase Chain Reaction (ASO-PCR) assay for screening the mutant allele at the three codons 351, 311 and 315 of the BCR-ABL ATP binding domain in Imatinib resistant CML patients. The phenotype of fifteen CML patients were successfully analyzed, thirteen patients were in complete cytogenetic response state (Philadelphia 0%) only two of those patients showed the Philadelphia chromosome. Three point mutations T1052C, T932C and C944T were identified by allele specific oligonucleotide polymerase chain reaction (ASO-PCR). One patient showed T1052C mutation, T932C was detected in three patients and two patients showed the C944T ATP binding domain mutation. In conclusion, conventional cytogenetic and molecular genetic tests are complementary techniques to show the exact types of abnormalities, which allow better evaluation of the genomic aberration involved in CML patients, specifically for kinase domain mutation that plays an important role in different diagnosis, prognosis, therapy treatment and drug resistant management of chronic myeloid leukemia.

N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic messenger RNA (mRNA). The total amount of m6A can be detected by several methods, such as dot blot analysis using specific m6A antibodies and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) ( Fu et al., 2014 ; Shen et al., 2016 ). Here we describe the method for fast detection of total m6A levels in mRNA by dot blot analysis using a specific m6A antibody.

Early methods for the detection of single-nucleotide polymorphisms (SNPs) were based on restriction fragment length polymorphism analysis (1) and the effects of base-pair changes on DNA fragment melting temperatures (denaturing gradient gel electrophoresis) (2). More recently, detection techniques have utilized allele-specific oligonucleotide (ASO) hybridization (3)(4), the single-stranded conformational polymorphism method (5), allele-specific priming of PCR (6)(7), primer-guided nucleotide incorporation assays (8), and oligonucleotide ligation assays (9). The technique of differential hybridization to ASO probes has been widely used. Under high stringency conditions, synthetic DNA probes will only anneal to their complementary target sequences in the sample DNA if they are perfectly matched, with a single base-pair mismatch sufficient to prevent formation of a stable probe-target duplex (10)(11). PCR-amplified genomic DNA products are applied to nylon filters as a series of “dot blots” to which radiolabeled ASOs are hybridized (4). The reverse approach can also be used with ASO probes fixed to a membrane support (12) or in a microtiter plate format (13). A combination of PCR amplification with allele-specific hybridization in a microtiter plate format using colorimetric ELISA-based detection was developed (PCR-ELISA ASO typing) that provided highly sensitive and quantitative detection; this method was initially applied to HLA typing (14)(15). The PCR-ELISA ASO typing method amplifies the number of copies of a DNA segment from a sample of genomic DNA by PCR with the incorporation of digoxigenin-11-dUTP. Samples are analyzed in a microtiter plate format by alkaline denaturation and hybridization to biotinylated allele-specific capture probes bound to streptavidin-coated plates. The hybridized DNA is detected by ELISA, using anti-digoxigenin horseradish peroxidase conjugate and the colorimetric substrate 2,2′-azino-di-(3-ethylbenzthiazolinsulfonate) (14). Detection of SNPs by allele-specific PCR, also known as the amplification refractory mutation system (ARMS), utilizes a primer with a 3′ mismatch at …

Within the past few years there has been increasing evidence that the genetic variation in the genes coding pro- and anti-inflammatory markers may play an important role in the pathogenesis of various human diseases, including stroke. The aim of the study was to evaluate the association of Interleukin-10 (IL-10)-1082 G/A, promoter polymorphism (rs1800896) with ischemic stroke in a South Indian population from Andhra Pradesh. In this study 480 ischemic stroke patients and 470 age and sex matched healthy controls were included. The ischemic stroke patients were classified according to TOAST classification. The region of interest in the IL-10 gene was amplified by polymerase chain reaction with the use of allele specific oligonucleotide primers flanking the polymorphic region. Association between genotypes and stroke was examined by Odds Ratio (OR) with 95% confidence interval (CI) and Chi-square analysis. Significant difference was observed between the patients and healthy controls, in genotypic distribution as well as allelic frequency (p0.05). Multiple logistic regression analysis with forward stepwise selection using the potential confounders (sex, age, diabetes, hypertension, smoking and alcoholism) and IL-10 gene variant revealed that -1082 G/A polymorphism in the promoter region of IL-10 gene is significantly [adjusted OR=2.26; 95% C.I. (1.24-4.15), p0.001] associated with ischemic stroke in the South Indian population from Andhra Pradesh. We found significant association of this polymorphism with stroke of undetermined etiology (p0.001). Moreover, hypertensive and diabetic individuals bearing A allele of IL-10 gene in high frequency were found to be more predisposed to stroke.

Introduction: BCR-ABL kinase domain mutations represent the most important disease-related factor in chronic myelogenous leukemia (CML) resistance. Highly resistant clones may pre-exist and emerge rapidly. Patients with CML can acquire more than one BCR-ABL1 mutation, which may result in increased oncogenicity. Materials and Methods: Retrospective analysis of 50 patients of imatinib resistance was done in GCRI, from January 2014 to May 2014. Response to imatinib was defined according to the European LeukemiaNet 2009 criteria. Allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR) was performed on genomic DNA, extracted from peripheral blood mononuclear cells. Results: Average age was 40.75 years, 33 were males and 17 females. 47 (94%) were in chronic phase, 2 (4%) in accelerated phase, and 1 (2%) in blastic crisis. 29/50 were having low EUTOS score, whereas SOKAL score was low in 20, intermediate in 21 while only 9 had high SOKAL at presentation. Median duration of imatinib was 48 months. 43/50 had one or more than 1 mutation, T315I mutation in 5 (10%) patients, and M351T in 32% (16/50). Conclusion: The presence of M351T mutation in mutant clone leads to the development of T315I mutations development, and the detection of M351T mutation in the initial months of the therapy has a prognostic significance. ASO-PCR is more sensitive method of the detection of such mutations as compared to direct sequencing. We report low cytogenetic response (25%) and durability of response to 600 mg of imatinib, even in M351T mutation, after 400 mg of imatinib for median period of 4 years.

Reverse dot-blot is an effective method for detecting beta-thalassemia (beta-thal) mutations. In this study, we report the cause of a misdiagnosis by reverse dot-blot. One patient with a beta-globin genotype that was initially diagnosed as a codon 17 (A>T) homozygote by reverse dot-blot at our Clinic, was later shown to be a codon 17 (A>T) heterozygote by direct sequencing of the amplified fragment and by sequence analysis of a recombinant T vector plasmid containing an amplified fragment of the beta-globin gene. A polymorphic locus around the mutation site within the haploid DNA that lacked the codon 17 mutation was identified. This result suggests that when reverse dot-blot is used for the genetic diagnosis of beta-globin, polymorphic loci around the mutation site should be taken into consideration, and more allele specific oligonucleotide probes should be designed according to the polymorphic loci.

A method was developed for rapid identification of Dendrobium species by a dot blot hybridization assay. The dot blot system is based on species-specific amplified fragments derived from the internal transcribed spacer (ITS) region of different Dendrobium species as target DNA, which were blotted as dots on the nylon membrane. A small aliquot of the ITS amplified product from a selected species or mixed ITS DNA from several species was labeled by peroxidase and used as a probe for hybridization to the dot blot membrane. Simply visualizing the positive hybridization reaction between the probe DNA and the target DNA could identify the selected species. The D. officinal-specific and D. nobile-specific ITS-based probes could separately identify D. officinal and D. nobile. Eight Dendrobium species, including D. salaccense, D. brymerianum, D. ellipsophyllum, D. chrysanthum, D. officinale, D. thyrsiflorum, D. nobile and D. chrysotoxum could be simultaneously detected using their complex ITS amplified products as a probe, and the ITS DNA probe amount for detection by the dot blot membrane is very small, just 2.5 ng. The assay can also be used to identify the resource species of Fengdou Shihu and Huangcao Shihu from the commercial market. The identification based on the ITS sequence was superior to the conventional approaches in speed, sensitivity, and specificity. Therefore, the polymerase chain reaction (PCR)-dot blot hybridization assay can be considered a reliable method for general identification of commercial Shihu.

The mechanisms of aberrant expression of blood-group antigens are not clear in all cases. The aim of this work was to investigate Lewis blood type antigens and secretor status using biochemical and molecular biological methods in patients with oral lesions. We studied 148 subjects, half of whom suffered from oral lesions (benign, pre-cancerous and cancerous), while the other half were the healthy control group. We also investigated the Lewis phenotypes of fresh blood samples with a hemagglutination method. We analyzed polymorphisms of the FUT2 gene by ASOPCR (allele specific oligonucleotide– polymerase chain reaction) with specific primers for the G428 allele and the wild type allele of FUT2 gene. The FUT2 gene encodes the γ(1,2) fucosyltransferase (Se enzyme) that regulates expression of ABH antigens in the gastrointestinal tract and secretions. We found a higher intensity of oral disease in the non-secretor group (OR = 3.44). Fifty one % of the patients with oral pre-cancerous and cancerous lesions were non-secretors (Le a+ b−), in contrast with the healthy population. We observed a marginal association between secretor status and these lesions. Our study suggests that the lack of wild type FUT2 gene and a nonsecretor status appear to be an associated risk marker for the development of oral cancer in patients with oral lesions.

Single point mutations in the genes coding for hemostatic factors were shown to be major inherited predisposing factors for venous thromboembolism. However, their contribution in the development of non-diabetic coronary artery disease [nDCAD] remains controversial. Angiographically demonstrated nDCAD patients (n = 86) and healthy controls (n = 90) were included in the study. Genotype analysis of hemostatic gene polymorphisms were assessed by using CVD strip assay, based on allele specific oligonucleotide probes. The carrier frequency of factor V (FV) H1299R, prothrombin G20210A, glycoprotein (Gp) IIIa L33P, plasminogen activator inhibitor-I (PAI-1) 4G/5G, 4G/4G, 5G/5G, methylenetetrahydrofolate reductase (MTHFR) A1298C and beta-fibrinogen -455 G > A were similar between patients and controls. In contrast, frequency of FV Leiden was significantly higher among patients (12.5%) than controls (5%, OR: 7.94; 95%CI: 1.9-49.6) and FXIII V34L was significantly lower among patients (23.7%) than controls (40%, OR: 0.24; 95%CI: 0.1-0.89). In addition, the frequency of the MTHFR C677T polymorphism was 32.5% among patients compared with 42.5% in controls, of which the T/T genotype was significantly lower among patients (5%) than controls (17.5%, OR: 0.06; 95%CI: 0.01-0.58). No difference was observed in prevalence of prothrombin G20210A, FV H1299R, Gp IIIa L33P, PAI-1 4G5G, MTHFR A1298C, beta fibrinogen 455 G > A mutations between patients and controls. However, lower frequency of FXIII Val34Leu and MTHFR C677T polymorphisms may decrease, while FV Leiden polymorphism may increase development of nDCAD.

Isoelectrofocusing and PCR Amplification-Reverse Hybridization Assay in Evaluation of Alpha-1-Antitrypsin DeficiencyAlpha-1-antitrypsin deficiency is a potentially lethal genetic disorder, which has pulmonary and liver manifestations. The standardized biochemical and molecular diagnostic protocol for detection of clinically relevant alleles is needed. The paper summarizes current concepts about AATD, describes the potentials of isoelectric focusing and PCR amplification-reverse allele specific oligonucleotide hybridization assay in the detection of affected individuals and shortly presents our experiences in the evaluation of AATD. We conclude that the systematic clinical laboratory approach to AATD might be based on the combination of mentioned methods, coordinated by alpha-1-antritrypsin quantification. Additionally, its complete medical implementation is achieved through teamwork between clinical chemists, molecular biologists and clinicians.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited disorder worldwide including southern China. We developed and validated a reverse dot blot (RDB) assay for the rapid and simultaneous genotyping of six mutations (c.95A>G, c.871G>A, c.1004C>T, c.1024C>T, c.1376G>T and c.1388G>A), that were common mutations in the Chinese G6PD deficiency population, and one polymorphism (c.1311C>T). Reliable genotyping of wild-type and mutant genomic DNA samples was achieved by means of a test strip onto which allele-specific oligonucleotide probe lines are fixed in parallel. This method involves a multiplex PCR amplification of three fragments in the G6PD target sequence and a manual hybridization/detection protocol. The entire procedure starting from blood sampling to the identification of mutations requires less than 6 h. The diagnostic reliability of this reverse dot blot assay was evaluated on 207 pre-typed samples by using direct DNA sequence analysis in a blind study. The reverse dot blot typing was in complete concordance with the reference method. The reverse dot blot assay was proved to be a simple, rapid, highly accurate, and cost-effective method to identify common G6PD mutations in Chinese population.

Frogeye leaf spot (FLS), caused by Cercospora sojina Hara, has become a more frequent disease in soybean, Glycine max (L.) Merr., across the USA. The Rcs3 gene provides resistance to all known races of C. sojina. To provide resources for efficient marker-assisted selection (MAS) of Rcs3, we developed and evaluated several single nucleotide polymorphisms (SNP) and insertions or deletions (InDels) markers linked to Rcs3. We surveyed 13 bacterial artificial chromosome (BAC) end sequences that were anchored with simple sequence repeat (SSR) markers Satt244 and Satt547 along with the two SSRs containing clones in six soybean cultivars (Davis, Cook, and Young that have the Rcs3 allele and Blackhawk, Bragg, and Lee that have the rcs3 allele) for SNPs. A total of 19 SNPs/ InDels were identified and validated, but only 11 mapped near Rcs3 in a F 2 population of Davis × Blackhawk. The Rcs3 gene was positioned near Satt244 and 0.50 cM from SNPs AZ573TA150 and AZ573CA393. None of the SNPs or InDels identified appeared to contribute directly to the Rcs3 phenotype, but 11 mapped within a 3-cM interval surrounding Rcs3. These 11 markers were further validated for association with Rcs3 and for their potential in MAS in 64 lines and cultivars. Our results suggest that the two markers AZ573TA150 and AZ573CA393 could be used in MAS for Rcs3. Allele specific oligonucleotide probes specific for MAS were developed for a direct hybridization assay on a Luminex 100 flow cytometry platform.

A single-stranded DNA and its point mutant can be separated with affinity capillary electrophoresis (ACE) in which an appropriate ligand DNA is used as a pseudo-stationary affinity phase. In this paper, we systematically examine the effects of ligand sequence, capillary temperature, and cation concentration on the ACE separation quality, which is quantitatively evaluated in terms of resolution and peak height. For fine tuning of the affinity, control of MgCl2 concentration and insertion of a spacer sequence into the ligand DNA are more effective than control of the capillary temperature. For design of the ligand DNA, a simple strategy is proposed, based on the prediction of melting temperature. This strategy was tested with eleven different sample sequences. All of them were separated under the same conditions (250 μM MgCl2 and 25 °C), and nine cases of them had satisfactory separation qualities.

Purpose: The polymerase chain reaction is generally used for mutational analysis of the galactose-1-phosphate uridyl transferase (GALT) gene in the diagnosis of galactosemia. This method is problematic when used in families of Ashkenazi Jewish descent. Methods: We amplified the GALT gene from leukocyte DNA followed by allele specific oligonucleotide hybridization, DNA sequencing and Southern Blot analysis to determine the mutant alleles causing galactosemia in a representative Jewish family. Results: The proband's diagnosis of galactosemia was confirmed by high levels of erythrocyte galactose-1-phosphate, absence of erythrocyte GALT activity and impaired total body oxidation of galactose to expired CO2. Initial molecular analysis of GALT alleles in the family showed homozygosity for a K285N missense mutation in the proband, homozygosity for N314D in the mother and heterozygosity for N314D and K285N in the father. These results contradicted Mendelian logic. Southern blot hybridization with GALT cDNA proved the presence of a complex 5 kb GALT deletion in the proband and her mother's DNA enabling a corrected genotype. Conclusions: Since a deletion of the GALT gene is a common mutation causing galactosemia among Ashkenazim Jewish families, this deletion should be suspected and tested for by genomic hybridization or by using primers specific for the 5 kb deletion.

Contains fulltext : 50264tits.pdf (Publisher’s version ) (Closed access) OBJECTIVES: To develop and validate a novel genotyping approach, named infrared Fluorescence Allele Specific Hybridization (iFLASH), which combines the principles of allele specific oligonucleotide (ASO) hybridization with the advanced possibilities of infrared imaging. DESIGN AND METHODS: As an example, we genotyped the 55L > M and the 192Q > R common genetic variants of the paraoxonase-1 gene in 92 DNA samples using the iFLASH technique, and validated the outcomes with the restriction fragment length polymorphism (RFLP) and TAQman genotyping assays. RESULTS: There was a 100 percent agreement in genotype outcome among the three methods. CONCLUSIONS: Although we found complete unity in genotype outcome, the iFLASH assay has essential advantages over the RFLP and TAQman genotyping assays. First, the iFLASH technique is capable of handling up to 1536 samples per assay, which makes it a suitable technique for high-throughput genotyping. Secondly, because the costs per assay are lower, high-throughput genotyping with iFLASH is affordable.

A new concept of genetic field effect transistors (FET) is proposed for detection of specific binding of bio-molecules, which is in principle based on charge density change at the gate insulator. The electrical signals of the genetic FET were found to change after allele specific oligonucleotide hybridization, intercalation and primer extension. We demonstrated experimentally that the SNP genotyping could be achieved by the use of the genetic FETs in combination with allele specific extension reaction. Single base extension on the gate could be detected by the use of FET, which indicated possibility toward DNA sequencing. The genetic FET platform based on the direct transduction of oligonucleotide extension into electrical signal is suitable for a simple, accurate and inexpensive system for SNP typing in clinical diagnostics.

Contains fulltext : 48971.pdf (Publisher’s version ) (Closed access) BACKGROUND: Extrahepatic biliary stenosis (EBS) has malignant and benign causes. Patients with EBS are at risk of having or developing malignancy. Accurate diagnostic tests for early detection and surveillance are needed. The sensitivity of biliary cytology for malignancy is low. K-ras mutation analysis on brush cytology is a valuable adjunct, but specificity is low. A quantitative test for K-ras mutations has been developed: the amplification refractory mutation system (ARMS).AIM: To assess the test characteristics and additional value of ARMS in diagnosing the cause of EBS.METHODS: Brush samples from endoscopic retrograde cholangiopancreatography were collected from 312 patients with EBS. K-ras mutation analysis was performed using ARMS-allele specific amplification was coupled with real time fluorescent detection of PCR products. Results were compared with conventional cytology and K-ras mutation analysis using allele specific oligonucleotide (ASO) hybridisation, and evaluated in view of the final diagnosis.RESULTS: The test characteristics of ARMS and ASO largely agreed. Sensitivity for detecting malignancy was 49% and 42%, specificity 93% and 88%, and positive predictive value (PPV) 96% and 91%, respectively. The sensitivity of ARMS and cytology combined was 71%, and PPV was 93%. The specificity of ARMS could be increased to 100% by setting limits for the false positives, but reduced sensitivity from 49% to 43%.CONCLUSIONS: ARMS can be considered supplementary to conventional cytology, and comparable to ASO in diagnosing malignant EBS. A specificity of 100% can be achieved with ARMS, which should be considered in the surveillance of patients at risk for pancreatic cancer.

Development of specific DNA probes targeting rDNA internal transcribed spacer (ITS-1 or -2) sequences is described for the detection of strains representing uninucleate and binucleate Rhizoctonia spp. and Suillus bovinus. Discriminatory taxon/species-specific target sequences were identified following full length ITS sequence alignment of the test fungal sequences and those of other root associated pathogenic or mycorrhizal fungi. Both long (124–151 bp) and shorter (20–25 bp) probes were generated for assessment in Southern dot blot and liquid hybridization ITS capture-fragment length polymorphism assays. Fungal genomic DNA was presented as the target in dot blot protocols using the longer DIG (digoxigenin) labelled probes whilst the shorter 3′ biotin-labelled oligonucleotide probes were hybridized to PCR amplified full length ITS (ITS1-5.8S-ITS2) in both dot blot and liquid hybridization assays. The optimal hybridization temperatures for dot blot detection also produced maximal target specific signals in the liquid hybridization protocol. The latter protocol was found to be more discriminatory as target fungi were detected on the basis of combined probe hybridization-ITS capture and 5′ Cy-5 labelled ITS length polymorphism analysis (±5 bp) following denaturing sequencing gel electrophoresis in a ALFexpress DNA sequencer.

Background: Congenital cataract and microphthalmia are highly heterogeneous congenital eye disorders that affect normal vision. Although mutation in several genes has been shown to cause congenital cataract and microphthalmia, genetic studies associating single-nucleotide polymorphisms with these conditions is scarce. Hence, the present study aims to investigate the association of bone morphogenetic protein 4 (BMP4)-V152A (rs17563), and SIX homeobox 6 (SIX6)-H141N (rs33912345) polymorphisms with congenital cataract and microphthalmia in Western Indian cohorts. Materials and Methods: BMP4-V152A and SIX6-H141N were genotyped in 561 participants comprising of 242 congenital cataracts, 52 microphthalmia, and 267 controls using polymerase chain reaction (PCR) and allele specific oligonucleotide (ASO)-PCR method, respectively. Results: The frequency of BMP4- 152A was found to be significantly different between the cases and controls (Odds ratio (OR) 95% confidence interval [CI] = 1.4 [1.03–1.76], P = 0.0275). The frequency of BMP4- 152AA genotype was found to be significantly higher in congenital cataract cases as compared to controls (OR [95% CI] = 2.1 [1.14–3.67], P = 0.0154. The V-N haplotype of BMP4-V152A and SIX6-H141N was found to have a protective effect toward congenital cataract (OR [95% CI] = 0.72 [0.56–0.94], P = 0.0163) and microphthalmia (OR [95% CI] = 0.63 [0.40–1.01, P = 0.0541). Conclusions: The BMP4- 152AA genotype might play role in the causation of congenital cataract, whereas BMP4-SIX6 V-N haplotype might play a protective role toward the development of congenital cataract and microphthalmia.

We developed an assay using a real-time quantitative polymerase chain reaction (RQ-PCR) for the quantitative assessment of minimal residual disease (MRD) in childhood lymphoid malignancies by using a consensus V-region probe combining a allele-specific oligonucleotide (ASO) reverse primer. Our strategy employs a set consisting of a consensus V-region probe, an ASO reverse primer, and a patient-specific forward primer for clonal antigen-receptor (IgH, immunoglobulin heavy chain; TCR, T-cell receptor) gene rearrangements (IgH-ASO and TCR-ASO RQ-PCR assays). The limit of detection in both assays was 5 copies of the target/105 cell equivalents. We tested the assays in 17 childhood malignancies (14 cases of acute lym-phoblastic leukemia and 3 of non-Hodgkin’s lymphoma). High correlation coefficients of the standard curves (>0.980) and PCR efficiency (>0.95) were achieved with all primer/probe sets. In 2 (12%) of the 17 patients, ASO primers could not be designed because there was no junctional N-sequence. The quantitative data suggest that the copy number of clonal antigen receptors markedly decreased after induction therapy in 15 of 17 patients and that 1 patient relapsed and died of the disease. Consensus probes make it possible to examine a large number of patients with only a limited number of probes. The strategy used for IgH-ASO and TCR-ASO RQ-PCR assays is accurate and reliable in the clinical prospective study of MRD in childhood lymphoid malignancies.

Reverse line blot (RLB) hybridization has become a well-established and widely used method for the multiplex identification of several Babesia and Theileria species in hosts and tick vectors. The procedure is based on the simultaneous PCR amplification of a polymorphic region of the 18S rRNA gene from different piroplasms followed by identification of the individual species by hybridization to species-specific oligonucleotide probes covalently linked to a nylon membrane in a macroarray format.

MYD88 L265P mutation has been reported in ∼90% of Waldenstrom's Macroglobulinemia (WM) patients and immunoglobulin M (IgM) monoclonal gammopathies of uncertain significance (MGUS), as well as in some cases of lymphoma and chronic lymphocytic leukemia. The present study aimed to develop a real-time allele-specific oligonucleotide PCR (ASO-RQ-PCR) to detect the MYD88 L265P mutation. We first evaluated the reproducibility and sensitivity of the technique with a diluting experiment of a previously known positive sample. Then, we evaluated the applicability of the methodology by analyzing 30 selected patients (10 asymptomatic WM, 10 symptomatic WM, and 10 IgM MGUS) as well as 10 healthy donors. The quantitative ASO-PCR assay could detect the MYD88 L265P mutation at a dilution of 0.25%, showing an inverse correlation between the tumor cell percentage and the cycle threshold (CT) value, thus allowing for tumor burden quantitation. In addition, mutated cases were distinguished from the unmutated by >10 cycles of difference between CTs. To sum up, ASO-RQ-PCR is an inexpensive, robust, and optimized method for the detection of MYD88 L265P mutation, which could be considered as a useful molecular tool during the diagnostic work-up of B-cell lymphoproliferative disorders.

Developing a methylation-specific dot blot assay (MSP-DB) to increase the sensitivity and specificity of simultaneous methylation analysis of multiple genes is the goal of the present study to evaluate the methylation status of GSTP1 and RASSF1A from prostate cancer in Vietnamese males.The methylation of GSTP1 and RASSF1A was investigated by using the MSP in 50 prostate cancer and 17 benign prostate hyperplasia specimens. The MSP-DB assay that uses a single or multiple probes specifically detected the methylation status of a particular gene or of the two genes GSTP1 and RASSF1A at the same time in a series of samples. The sensitivity and specificity of the MSP-DB were compared to those of the MSP.The probes specifically hybridized with the methylated targets only in the MSP-DB, which allowed detecting GSTP1 and RASSF1A methylation in 23 of 50 and 14 of 50 patients with prostate cancer and in 2 of 17 and 4 of 17 patients with benign prostate hyperplasia. MSP-DB following the MSP assay improved the sensitivity of detection to more than 0.01 % methylated status of a given high CpG-rich region. One methylated MSP product corresponding to the GSTP1, lack of methylated cytosine, was clearly detected on gel electrophoresis but barely visible on MSP-DB. Thus, the MSP-DB is suitable to eliminate the risk of false-positive results.The MSP-DB dispels the weakness of MSP and increases the sensitivity to simultaneous methylation analysis of multiple genes. The MSP-DB is advantageous for the promotion of DNA methylation markers in progressing quickly toward clinical application.

Five polymerase chain reaction (PCR) products which could not be reliably typed by allele-specific oligonucleotide (ASO) probing at the human leukocyte antigen (HLA) DQA1 locus were analyzed by polyacrylamide gel electrophoresis and direct sequencing. The first method revealed the preferential amplification of only one of the two alleles in two cases. Direct sequencing of PCR products allowed unambiguous genetic typing but a high number of artifacts was observed. Several of these artifacts occurred in the sequences recognized by the ASOs. This finding provides an explanation for the mistyping in the ASO probing procedure because Taq polymerase errors both created new genetic specificities and eliminated site-specific polymorphisms. Reversed-phase HPLC-MS of the five forensic templates showed a high degree of DNA damage. These data together indicate that the risk of mistyping when using the ASO probing procedure cannot be neglected in the forensic analysis of damaged DNA samples.

The polymerase chain reaction (PCR) was used for genetic characterization of 45 samples taken from the city of Elaziğ in Turkey. The polymorphism at the human leukocyte antigen DQalpha locus was detected. Allele and genotype frequencies were determined for unrelated individuals at this locus. Laboratory analyses were done by PCR amplification of DNA. Hybridization to allele specific oligonucleotide probes was performed using a reversed dot-blot typing method. The collected genotype and allele frequencies have been tested, and a comparison was made with other population surveys of this locus. Allele frequencies ranged from 3.3% (allele 1.3) to 36.7% (allele 4), with a discrimination power of 0.92. No deviation was seen from Hardy-Weinberg equilibrium in the findings.

Large-scale clinical studies on detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) have shown that quantification of MRD levels is needed for reliable MRD-based risk group classification. Recently, we have shown that 'real-time' quantitative PCR (RQ-PCR) can be applied for this purpose using patient-specific immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements as PCR targets with TaqMan probes at the position of the junctional region and two germline primers. Now, we tested an alternative approach on 35 immunoglobulin heavy chain (IGH) gene rearrangements, by designing three germline JH TaqMan probes to be used in combination with one of six corresponding germline JH primers and one allele specific oligonucleotide (ASO) primer complementary to the junctional region. In nine cases in which both approaches were compared, at least similar (n = 4) or slightly higher (n= 5) maximal sensitivities were obtained using an ASO primer. The ASO primer approach reached maximal sensitivities of at least 10(-4) in 33 out of 35 IGH rearrangements. The reproducible range for accurate quantification spanned four to five orders of magnitude in 31 out of 35 cases. In 13 out of 35 rearrangements the stringency of PCR conditions had to be increased to remove or diminish background signals; this only concerned the frequently occurring JH4, JH5 and JH6 gene rearrangements. After optimization of the conditions (mainly by increasing the annealing temperature), only occasional aspecific amplification signals were observed at high threshold cycle (CT) values above 42 cycles and at least six cycles above the CT value of the detection limit. Hence, these rare aspecific signals could be easily discriminated from specific signals. We conclude that the here presented set of three germline JH Taq-Man probes and six corresponding germline JH primers can be used to develop patient-specific RQ-PCR assays, which allow accurate and sensitive MRD analysis in almost all IGH gene rearrangements. These results will facilitate standardized RQ-PCR analysis for MRD detection in large clinical studies.

Drugs and carcinogens are excreted from the body after metabolic conversion involving enzymes mediating oxidative metabolism and conjugation. Many of the corresponding genes exhibit functional polymorphisms that contribute to individual cancer susceptibility. To increase the efficiency and to facilitate genotyping, we developed a combined approach (PCR-ASO) which includes multiplex PCR and allele-specific oligonucleotide (ASO) hybridization. PCR primer pairs were used to amplify the following alleles/variants: CYP1A1*1, *2A, *2B; CYP2D6*3, *4; NAT1*4, *3, *10, *11, *14, *15; and NAT2*4, *5A, *5B, *5C, *6A, *7B. The products were dot-blotted and polymorphisms were detected by hybridization with ASO probes for both wild-type and variant sites in parallel. This approach was validated by genotyping DNA samples from a French-Canadian population that was previously analyzed by PCR-RFLP. The variants frequencies were compared with the data on other populations available in the literature. The PCR-ASO assay appears to be simple, efficient, and cost-effective, particularly if a large number of samples are to be screened for several DNA variants. This approach has potential for automation with microplates and robotic workstations for high throughput.

Background: Although germline mutations in BRCA1 play a central role in familial breast and ovarian cancers, to date, no somatic mutations in BRCA1 have been reported in sporadic breast cancer, and only five somatic mutations have been identified in the sporadic ovarian carcinomas. Because loss of heterozygosity appears frequently at the BRCA1 locus in nonfamilial breast and ovarian carcinomas, we searched for mutations in the BRCA1 gene in sporadic ovarian tumors. Methods: We developed a detection system based on PCR and reverse allele-specific oligonucleotide hybridization on membrane strips for the simultaneous detection of 17 frequently occurring mutations in the BRCA1 gene. Results: As little as 2% mutant DNA in a sample could be detected. Two of 122 DNA samples isolated from sporadic ovarian tumor biopsies contained the Cys61Gly mutation. Both mutations were germline mutations. One of these was an ovarian metastasis of a primary fallopian tube carcinoma. The tubal carcinoma was also confirmed to contain the Cys61Gly mutation. Conclusions: This is the first report that a germline BRCA1 mutation is associated with primary tubal carcinoma. The 17 specific mutations in the BRCA1 gene do not play a major role in the tumorigenesis and progression of sporadic ovarian cancer.

Objectives: To implement cost effective and clinically relevant thrombophilic genotyping and homocysteine analysis in our coagulation laboratory. Methods: We describe genotyping assays for three of the genetic defects associated with hereditary thrombosis: factor V Leiden A1691G, methylenetetrahydrofolate reductase C677T, and prothrombin gene G20210A. A second confirmatory assay for factor V Leiden using allele specific oligonucleotide polymerase chain reaction is also presented. We suggest an algorithm for the rational integration of the traditional assays routinely used to investigate venous thrombosis with genotyping and plasma homocysteine measurements. Results: These polymerase chain reaction based assays were designed to be performed under identical reaction conditions, permitting simultaneous setup, amplification, digestion, and analysis. Conclusions: The three genotyping assays presented are robust and relatively easy to perform. Use of an algorithm will ensure efficient resource utilization and minimize unnecessary testing.

An assay is described which is useful for genetic screening of the two most prevalent mutations that cause hereditary fructose intolerance (HFI). Both mutations lie within exon 5 of the aldolase B gene. Amplification of exon 5 from genomic DNA isolated from peripheral lymphocytes using biotinylated aldolase B-specific primers yields a biotin-tagged probe. This probe is hybridized to complementary poly(dT)-tailed allele specific oligonucleotides (ASOs) that are bound to a nylon membrane. The length of the ASOs, the amount bound to the membrane and the time of hybridization are optimized for discrimination of all four alleles under the same hybridization conditions. Detection of biotinylated amplified DNA is performed by creating an avidin-alkaline phosphatase complex and visualization by chemiluminescence. This assay can rapidly detect the two mutations, A149P and A174D, which cause >70% of HFI worldwide, and offers a rapid and sensitive assay that is much less invasive for the diagnosis of this often difficult to diagnose disorder.

Sixth-five 14-thalassaemia genes from 14 unrelated Chinese β-thalassaemia major patients and 37 Chinese β-carriers were analysed by allele-specific oligonucleotide (ASO) hybridization after DNA amplification by the polymerase chain reaction (PGR). Six mutations were studied and are represented by 49.2% of codon 41-42, 30.8% of IVSII #654, 6.2% of 17β 3.1% of IVSI #5 (G→G) and 1.5% of -28 TATA box. The complete mutations responsible for β-thalassaemia major in 13 of our 14 affected families were identified. For these families prenatal diagnosis at 10 weeks gestation using DNA amplification and ASO hybridization will replace the globin chain biosynthesis technique at 19 weeks gestation

The most common form of carnitine palmitoyltransferase II (CPT II) deficiency occurs in adults and is characterized by muscle pain, stiffness, and myoglobinuria, triggered by exercise, fasting, or other metabolic stress. This study reports the molecular heterogeneity of CPT2 mutations and their biochemical consequences among a series of 59 individuals who were suspected of having CPT II deficiency based on the decreased CPT activity observed in muscle or leukocytes samples, clinical findings, or referral for mutation analysis from other laboratories. Only 19 subjects were considered to be at particularly high risk of CPT II deficiency based on review of their clinical symptoms and residual CPT activity. The samples were initially screened for 11 mutations with allele-specific oligonucleotides (ASO). Extensive sequence analysis was subsequently performed on 14 samples which either had a CPT2 mutation detected by ASO screening or the residual CPT activity was below that observed in ASO positive samples. Three known (P50H, S113L, and F448L) and three novel mutations were identified among 13 individuals in this study. A single nucleotide polymorphism was also identified 11 bp distal to the CPT2 polyadenylation site that will be useful for linkage analysis. Two of the new mutations were single nucleotide missense mutations, R503C and G549D, that occurred in highly conserved regions of the CPT isoforms, and the third was a frameshift mutation, 413 delAG, caused by a 2-bp deletion upstream of a previously identified missense mutation, F448L. The 413 delAG mutation was the second most common mutation identified in our study (20% of mutant alleles) and all individuals with the mutation were of Ashkenazi Jewish ancestry suggesting a defined ethnic origin for the mutation. Despite rigorous mutation analysis, six of 13 individuals identified with CPT2 mutations remained as heterozygotes. We propose that heterozygosity for certain CPT2 mutations, S113L and R503C, is sufficient to render individuals at risk of clinical symptoms. Hum Mutat 13:210–220, 1999. © 1999 Wiley-Liss, Inc.