Specific Oligonucleotide Polymerase Chain Reaction Research Articles

Genotyping of blood groups in alloimmunized patients with β-thalassemia major by T-ARMS-PCR and multiplex-aso-pcr

IntroductionBeta-thalassemia major is a severe hemolytic anemia requiring life-long blood transfusion. Planned random donor blood transfusion is associated with alloimmunization against incompatible antigens. Determination of the minor blood group systems phenotype or genotype, and administration of the compatible blood components can significantly reduce the rate of alloimmunization.The present study aimed to determine the prevalence of alloimmunization, and genotype/phenotype characteristics of the minor blood groups systems in patients with β-thalassemia major. Material and methodsThis study was conducted on 1147 β-thalassemia major patients. Initially, antibody screening and antibody identification were performed. Then, phenotyping and genotyping for the Rh, Kell, Kidd, and Duffy blood groups were done in alloimmunized patients using monoclonal antibodies and Multiplex-Allele Specific Oligonucleotide-Polymerase Chain Reaction (Multiplex-ASO-PCR) and Tetra-primer amplification refractory mutation system–PCR (T-ARMS-PCR), respectively. Any phenotype/genotype discrepancy was assessed by direct sequencing. ResultsNinety-seven (8.5 %) out of 1147 patients had alloantibodies against the minor blood group antigens (44 males, 45.4 %, and 53 female, 54.6 %). The most common alloantibodies were against the RH (n: 47, 48.5 %), and the Kell (n: 23, 23.7 %) blood groups systems. Twenty-three (2.1 %) genotype/phenotype discrepancies out of 1067 tests, including 9 in the Rh (9.3 %), 8 in Duffy (34.8 %), and 6 in Kidd (26.1 %) blood groups were detected. No discrepancy was found in the Kell blood group system. Direct sequencing revealed that the results of molecular methods were correct. ConclusionMultiplex-ASO-PCR and T-ARMS-PCR molecular methods are fast, reliable and cost-benefit molecular methods for the minor blood group genotyping in multi-transfused β-thalassemia major patients.

Genetic Polymorphism in TNF-α-308 G/A and TNF-β +252 A/G, as Prognostic Biomarker in Breast Cancer Patients among Indian Population.

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<0.0001). However, no association was seen in TNF-β +252 A/G both at genotypic and allelic level. The GG genotype of TNF-β +252A/G is higher in grades III (p<0.01) patients. Conclusion:Our results suggest that TNF-α-308G/A polymorphism showed significant association with breast cancer patients.

Cytogenetic and molecular genetic studies of number of chronic mylogenous leukemia in Erbil province

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.

Expression of the Gene Encoding Secretor Type Galactoside 2 &amp;lt;i&amp;gt;α&amp;lt;/i&amp;gt; Fucosyltransferase (&amp;lt;i&amp;gt;FUT&amp;lt;/i&amp;gt;2) and CD44 Marker in Urogenital Tumors

The aim of this work was to investigate the FUT2 gene, the secretor status and the expression of CD44 protein in epithelial cells obtain from saliva and urine samples from patients with urogenital tumours. We studied 104 subjects. Half of them had urogenital tumours, while the other half was the healthy control group. We determined the secretor status in saliva with the haemagglutination inhibition technique. We analyzed the FUT2 polymorphism by allele specific oligonucleotide–polymerase chain reaction (ASO-PCR) with specific primers for the G428 allele and the wild type allele of FUT2 gene. We found higher intensity of urogenital disease in the non-secretor group (OR = 3.44). In contrast with the healthy population, 51.6% of the patients with potential malignant urogenital lesions and cancerous lesions were non-secretors. We also investigate by confocal microscopy, the expression of CD44 protein in epithelial cells obtained from urine samples from these patients. The results obtained also showed fluorescence corresponding to the presence of CD44 protein in samples from patients diagnosed with cancer. 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 urogenital tumors. Several mechanisms, based on the properties of CD44 as the major hyaluronan CD44 in cell carcinomas receptor, have been proposed to explain the role of elevated CD44 expression during tumour development and progression. CD44 might be a good candidate as a predictor of prognosis in this group of cancers.

Significance of Minimal Residual Disease in Chronic Lymphocytic Leukemia

Newly introduced highly effective treatment options increase the importance of minimal residual disease measurement in chronic lymphocytic leukemia. Minimal residual disease is gaining interest mainly as a predictive marker; however, clinical significance of minimal residual dis-ease in chronic lymphocytic leukemia in many different situations remains unresolved. Factors with a possible impact on the clinical significance of minimal residual disease are as follows: technique for minimal residual disease quantification, treatment regimen, peripheral blood vs. bone marrow analysis or time -point for sampling. Highly sensitive methods now available to evaluate minimal residual disease can detect a single chronic lymphocytic leukemia cell in 10(-4)- 10(-5) leukocytes using either allele -specific oligonucleotide polymerase chain reaction or multicolor flow cytometry. Minimal residual disease quantification as a surrogate marker to assess treatment efficacy in routine hematological practice has to be further evaluated.

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Objectives VEGF is key component in the differentiation, invasion, regulation of vascular remodeling and the survival of cytotrophoblasts. In preeclampsia expression of cytotrophoblasts is disregulated. Evidence supports the hypothesis that VEGF plays an important role in the pathogenesis of preeclampsia. In this study, we aimed to determine whether VEGF genetic polymorphisms are associated with risk of early onset of severe preeclampsia. Methods A cross-sectional study with observational analytic was conducted in 32 pregnant women with early onset severe preeclampsia and 32 normal pregnant women. After peripheral blood was obtained from all women and genomic DNA was isolated, we genotyped +405G>C polymorphisms in the 5′- untranslated region and +936C>T polymorphisms in 3′-untranslated region of the VEGF gene genotype using Allelic Specific Oligonucleotide–Polymerase Chain Reaction (ASO–PCR) methods. Results The distribution of G allele of VEGF +405 more dominant (60.9%) than C allele (39.1%) in early onset severe preeclampsia. Conversely in normal pregnant women, the frequency of C allele of +405 (53.1%) little bit higher than G allele (46.9%). The distribution of the T allele of +936 approximately was the same between early onset severe preeclampsia (35.9%) and normal pregnant women (31.3%). Overall, the genotype and allele frequency distribution of the two polymorphisms (+405 and +936) were not significantly different between women with and without the disease ( p > 0.05). Conclusions There was no evidence of an association between VEGF allele or genotype frequencies and early onset preeclampsia in our study. VEGF gene polymorphisms studied are unlikely to be major predisposing for early onset severe preeclampsia in the Indonesian population. Disclosures M. Susanna: None. E.G. Dachlan: None.

Sensitive allele specific oligonucleotide-polymerase chain reaction in detection of preexisting mutations in imatinib-resistant chronic myelogenous leukemia patients: A retrospective analysis

Introduction: Pre-existing BCR-ABL kinase domain mutation leads to Imatinib resistance. Methods: Retrospective analysis of 50 patients of Imatinib resistance was done in GCRI, from January 2014 till May 2014. Allelle Specific Oligonucleotide–Polymerase Chain Reaction (ASO-PCR) was performed on Genomic DNA, of peripheral blood mononuclear cells (PBMCs). Results: 47 (94%) were in Chronic phase, 2 (4%) in accelerated phase, 1 (2%) in blastic crisis. Median duration of Imatinib was 48 months. 43/50 had one or more than 1 mutation, T315I mutation in 5 (10%) patients, M351T in 32% (16/50) and F311L in 8. Conclusion: We report low cytogenetic response (25%) and durability of response to 600 mg of Imatinib, even in M351T mutation.

A Novel Method for the Detection of Minimal Residual Disease in B-Cell Malignancies: Ion Semiconductor Sequencing for the Evaluation of Igh Gene Rearrangements

Background:Minimal residual disease (MRD) detection is of high clinical relevance in patients with B-cell malignancies and is generally a surrogate parameter to evaluate treatment response and long-term prognosis. IgH gene rearrangements can be used as molecular marker in approximately 80% of lymphoma and myeloma patients since they represent lineage-specific markers and the complementarity determining region 3 (CDR3) is unique to each clone. To date, allele specific oligonucleotide polymerase chain reaction (ASO-PCR) and real-time quantitative polymerase chain reaction (RQ-PCR) are considered the most sensitive and widely applicable methods for MRD detection. A major disadvantage of ASO-PCR and RQ-PCR assays, is the use of specific primers and probes for every individual patient. Clone-specific primers and probes are not only expensive but also time-consuming to design and to test, which limits their wide applicability in the clinical setting. The recent major improvements in next generation sequencing (NGS) technologies, provide the opportunity to identify and quantify clonotypes with consensus primers combining the benefits of high sensitivity and universal applicability. The present work was designed to overcome ASO-PCR and RQ-PCR limitations by developing a feasible method for rearranged IgH genes amplification, NGS and analysis using Ion Torrent Personal Genome Machine (IT-PGM).Methods:To define a multiplex PCR protocol, DNA from 7 CLL patients, previously shown to bare a family specific clonal VDJ rearrangement, was amplified with a pool of the seven different family-specific IgH-V primers, and a consensus JH primer (Voena et al., Leukemia 1997). After Sanger sequencing, results were compared to the ones obtained with singleplex PCR protocol. Once validated, the multiplex PCR protocol was used to amplify DNA from patients and serially diluted (up to 10-8 ) DNA from Namalwa cell line (bearing a known IgH rearrangement) and subsequently sequenced on the IT-PGM using the 316 Ion-chip. NGS data were analyzed by using the IMGT-High V-Quest web server tool and the statistical software R. RQ-PCR was used to quantify the specific VDJ rearrangement in the serially diluted Namalwa DNA solutions and in DNA from patients as previously described (Farina et al, Haematologica 2009). RQ-PCR data were analyzed through a relative quantification procedure.Results:The multiplex PCR reactions we have tested, demonstrated the same specificity as the standard singleplex PCR protocol and therefore was used to construct the DNA library required for IT-PGM-based sequencing. The IT-PGM sequencing output is represented by at least 400000 reads per sample with a minimum average coverage of the VDJ repertoire of 500x. The IMGT-High V-quest tool allows a user-friendly web based analysis and a deep molecular characterization of the IgH recombinatorial repertoire. Namalwa clonal CRD3 sequences were detected up to a dilution of 10-5 without the need for specific CDR3 primers. Comparability of NGS and ASO RQ-PCR results was assessed. The use of CDR3 specific primers, along with the specific IgH-V family fluorescent probe, enabled the identification of clonal VDJ rearrangements with a sensitivity up to 10-5 (2/3 replicates) and 10-6 (just 1/3 replicates) in Namalwa Cell Line. Similar results were obtained when we characterized the IgH recombination repertoire of two CLL patients over time.Conclusions:IgH sequencing with the IT-PGM platform showed at least the same level of sensitivity as ASO RQ-PCR, without the need for patient-specific reagents. It also allows specific and detailed molecular characterization of the clonal rearrangements and could be easily incorporated into clinical laboratories for routine testing of MRD in B-cell malignancies. DisclosuresNo relevant conflicts of interest to declare.

Kinetics Of Low-Level Mutant Clones Detected By Subcloning and Sequencing In Tyrosine Kinase Inhibitor Resistant CML

BackgroundBCR-ABL1 kinase domain (KD) point mutation causes resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML) patients through impaired binding of TKI to the target site. Recent studies have reported that multiple mutations detected in 2-9% of patients with imatinib (IM)-resistant CML were associated with poor response rate and survival outcomes. However, biological characteristics and dynamics of multiple low-level mutations are still not assessed with a quantitative serial follow-up data in the same populations. AimsThe aim of this study was to investigate biological characteristics and dynamics of low-level mutations in the serial samples from the patients carrying multiple mutations using subcloning and sequencing. MethodsSince 2002, 414 CML patients were screened for mutation analysis due to sign of resistance to TKIs including imatinib (IM), nilotinib (NIL), dasatinib (DAS), bosutinib (BOS), radotinib (RAD) or ponatinib (PON) at Seoul St Mary's Hospital using direct sequencing and allele specific oligonucleotide-polymerase chain reaction (ASO-PCR). Among them, 31 patients carried ≥ 2 BCR-ABL1 kinase domain mutations. We analyzed 137 samples from these 31 patients using subcloning and sequencing (in total, 2737 colonies were sequenced). By cloning and sequencing, two or more missense mutations present in the same clone were defined as compound mutation and co-existence of single missense mutations in the separated clones was defined as polyclonal mutation. Co-existence of single missense mutation and compound mutation harboring two or more missense mutations in the same clone was defined as mixed mutation. Missense mutations detected by direct sequencing are defined as predominant mutations. Missense mutations detected by cloning and sequencing but not by direct sequencing are defined as low-level mutations. ResultsIn a total of 2737 colonies from 137 samples, 1596 (58%) colonies harbored ≥ 2 missense mutations with a median 2 (range, 2 – 7) mutations, and 905 (33%) colonies with a single mutation and 236 (9%) colonies with wild type were observed.In 2737 colonies, 692 different low-level mutations were detected by cloning and sequencing but not by direct sequencing. Among them, M244V, G250E, Y253H, E279K, T315I, F317L, M351T, E355A, F359I, and F359V were detected by direct sequencing in the followed-up samples, and the others remain undetectable by direct sequencing. To address whether these low-level mutations were distinct in the patients with TKI resistance, we applied the cloning and sequencing to samples from 3 healthy controls and 3 patients with optimal response to IM. 38 different mutations were detected healthy controls. 52 different mutations were detected in optimal responders. S349P, N374S, E450G, and S485P were detected in both healthy controls and optimal responders. All mutations detected in optimal responders except 3 missense mutations (V335A, F382V, and A395S) were detected as low-level mutations in the 31 patients' cohort with TKI resistance. Of 38 different mutations detected in healthy control, 31 mutations except 7 missense mutations (Y264H, M343R, A350V, E373G, G298R, E462K, and T495K) were also detected as low-level mutations in the 31 patients' cohort. ConclusionsWe showed basic characteristics and dynamics of low-level mutations by subcloning and sequencing. Some of the low-level mutant clones harboring previously known (clinically common) TKI resistant mutations changes to the predominant in the followed-up samples. Except them, most low-level mutant clones were not detected repeatly and did not increase gradually in the serial samples, implying that they may not have clinical significance. Disclosures:No relevant conflicts of interest to declare.

Expression of the FUT2 gene and CD44 marker in patients with oral lesions

The aim of this work was to investigate the FUT 2 gene, the secretor status and the expression of CD44 protein in epithelial cells obtain from saliva samples from patients with oral lesions (benign, pre-cancerous and cancerous lesions, n=94). We analyzed polymorphisms of the FUT2 gene by allele specific oligonucleotide–polymerase chain reaction. The FUT2 gene encodes the α(1,2) fucosyltransferase (Se enzyme) that regulates the expression of ABH antigens in secretions. Generally speaking, being a non-secretor has several disadvantages with regard to metabolism and immune function. In this study, we found that oral pre-cancerous and cancerous lesions were increased among individuals with non-secretor status and nonsense mutation 428G→A. Fifty-one percent of the patients with oral pre-malignant and malignant lesions were non-secretors, in contrast with the healthy population (OR=3.44). We observed a marginal association between secretor status and these lesions. Our study suggests that the lack of wild type FUT2 gene and a non-secretor status appear to be an associated risk marker for the development of oral cancer in patients with oral lesions.One of the genes involved in tumour processes is CD44, which appears to be one of the most promising candidates as a cancer diagnosis marker. We investigated, using confocal microscopy, the expression of CD44 protein in epithelial cells obtained from saliva samples from patients with oral lesions. The results obtained showed fluorescence corresponding to the presence of CD44 protein in samples from patients diagnosed with cancer and pre-cancer. These findings indicate that overexpression of CD44 molecule analyzed could be considered as a marker of risk in individuals with oral lesions.

Abl Kinase Domain Mutations in Imatinib-treated Egyptian Patients with Chronic Myeloid Leukemia

Background: Point mutations within ABL kinase domain (AKD) of the BCR-ABL gene are the most common cause of resistance to Imatinib Mesylate (IM) treated Chronic Myeloid Leukemia (CML) patients. Objectives: To assess the frequency, type and impact of AKD mutations on prognosis in a cohort of Egyptian CML patients. Patients and methods: Serial measurements of BCR-ABL transcripts level in 175 IM treated CML patients were performed using real time quantitative polymerase chain reaction (RQ-PCR). Mutation screening was performed by allele specific oligonucleotide polymerase chain reaction (ASO-PCR) in 72 patients including all 42 non-optimal responders; 28 resistant patients, 18 suboptimal responders in addition to 30 patients randomly selected with stable/ decreasing transcript level representing an optimal responder category. Results: AKD mutations were detected in 16/28 resistant patients (57%) at time of >2-fold rise in BCR-ABL transcript and in none of the 44 optimal or suboptimal responders (0%) with decreasing or stable transcript levels. From 16 positive patients, P-loop mutations were detected in 9 patients; Q252H in 3 patients (19%), Y253H in 2 patients (12%), Y253F in 2 patients (12%) and E255K in 2 patients (12%). T315I was detected in 1/16 (6%) patient. Regarding non P-loop mutation; V299L was detected in one patient (6%), M351T in 4 patients (25%), F359V in 2 patients (12%). One patient had both Y253H and E255K mutations. Ten/16 (62%) patients carrying mutations experienced disease progression versus 1/56 (2%) in non mutation group (p=0.001). Median progression free survival (PFS) and overall survival (OS) of the mutation group was 13.5 months and 37.5 months, respectively versus 42.6 months in non mutation group (p=0.001). The estimated PFS and OS at 49 months in patients with mutations were 37.5% and 56.3% respectively versus 98.2% in non mutation carriers (p 2-folds in IM resistant patients may signal progression that implies testing for AKD mutations and early planning for second generation tyrosine kinase inhibitors (TKIs). P-loop mutations are significantly associated with advanced CML phases and poorer OS than non-P loop mutations. ASOPCR is a valuable tool for detection of mutations in countries where sequencing facilities are not available.

Analysis of Bcr‐Abl kinase domain mutations in Korean chronic myeloid leukaemia patients: poor clinical outcome of P‐loop and T315I mutation is disease phase dependent

Despite durable responses to imatinib in chronic myeloid leukaemia (CML), mutations in Bcr-Abl kinase domain (KD) are known to induce imatinib resistance and cause poor clinical outcome. We characterized Bcr-Abl KD mutations in 137 Korean CML patients with imatinib resistance (n = 111) or intolerance (n = 26) using allele specific oligonucleotide polymerase chain reaction (PCR) and direct sequencing. Seventy (51%) patients harboured 81 mutations of 20 different types with increasing prevalence in advanced phase. Nine (13%) patients had multiple mutations. No mutation was found in intolerant patients. T315I was the most common mutation and P-loop was the hottest spot in Bcr-Abl KD. Patients harbouring P-loop mutation, T315I, or multiple mutations showed poor overall survival and progression free survival compared with patients harbouring other mutations. Survival analysis according to disease phase of mutation being detected and type of mutations provided correlation between P-loop or T315I mutation and poor overall survival in blast crisis, but not in accelerated phase (AP) or chronic phase (CP), indicating poor clinical outcome of particular mutations depends on disease phase. CML patients with imatinib resistance showed high rate (63%) of mutations in Bcr-Abl KD and therefore CML patients who do not respond to imatinib should be the candidates for mutation screening as molecular monitoring.

Effect of Fas -670 A/G gene polymorphism on corneal allograft endothelial rejection.

Human cornea expresses functional Fas-ligand capable of killing Fas+ activated lymphocytes. Fas expression is partly regulated by -670 A/G polymorphism in the promoter region of Fas gene. The aim of the present study is to determine the association between Fas-670A/G polymorphism and survival of corneal transplantation. In 276 graft recipients who mainly underwent penetrating keratoplasty because of keratoconus, bullous keratopathy and corneal opacity, Fas -670 A/G polymorphism was determined by allele specific oligonucleotide polymerase chain reaction (ASO-PCR) techniques. There was no statistically significant relationship between Fas -670 A/G polymorphism and rejection episode (p=0.35). Moreover, the relationship between this polymorphism and rejection episode outcome (transplant recovery vs failure) was not statistically significant (p=0.13). The results of the present study show no significant correlation between corneal graft rejection, rejection recovery and Fas -670A/G gene polymorphism.

Characterization ofBacillusandPseudomonasstrains with suppressive traits isolated from tomato hydroponic-slow filtration unit

Bacillus and Pseudomonas spp. are known to be involved in plant pathogenic fungi elimination during the slow filtration process used in tomato soilless cultures. We isolated 6-8 strains of both Bacillus and Pseudomonas from the top, middle, and bottom sections of filters and identified them after 16S rDNA sequencing. Four Pseudomonas strains were identified as Pseudomonas fulva, 5 as Pseudomonas plecoglossicida, and 12 as Pseudomonas putida. The use of specific oligonucleotide polymerase chain reaction primer sets designed from gyrB gene sequences additionally permitted the identification of 17 Bacillus cereus and 3 Bacillus thuringiensis strains. Ribotyping with EcoRI pointed out an important polymorphism within Bacillus and Pseudomonas strains. Molecular characterization did not reveal a correlation between the location of isolates within the filter (top, middle, or bottom) and bacterial identification or riboclusters. Functional aspects assessed by community-level physiological profiling showed marked phenotypic differences between Pseudomonas communities isolated from the top and bottom filter layers; differences were lower between Bacillus communities of different layers and far less noticeable between mixed communities of Bacillus and Pseudomonas. These strains were tested for several suppressive activities. Conversely to most Bacillus, the majority of Pseudomonas strains were auxin producers and promoted the growth of tomato plantlet roots. On the other hand, only Bacillus strains displayed antagonistic activities by inhibiting the growth of pathogenic fungi frequently detected in soilless cultures. Siderophores were produced by nearly all bacteria, but at higher amounts by Pseudomonas than Bacillus strains. The biocontrol agent potentiality of certain strains to optimize the slow filtration process and to promote the suppressive potential of nutrient solution is discussed.

Genetic determinants of heritable venous thrombosis: genotyping methods for factor V LEIDEN A1691G, methylenetetrahydrofolate reductase C677T, prothrombin G20210A mutation, and algorithms for venous thrombosis investigations

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 LeidenA1691G, 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.