Biomedical subjects

B Gharizadeh

Publications and source records attributed to B Gharizadeh.

6 recordsLinked to original sources

PubMed2001

Genotyping by apyrase-mediated allele-specific extension.

This report describes a single-step extension approach suitable for high-throughput single-nucleotide polymorphism typing applications. The method relies on extension of paired allele-specific primers and we demonstrate that the reaction kinetics were slower for mismatched configurations compared with matched configurations. In our approach we employ apyrase, a nucleotide degrading enzyme, to allow accurate discrimination between matched and mismatched primer-template configurations. This apyrase-mediated allele-specific extension (AMASE) protocol allows incorporation of nucleotides when the reaction kinetics are fast (matched 3'-end primer) but degrades the nucleotides before extension when the reaction kinetics are slow (mismatched 3'-end primer). Thus, AMASE circumvents the major limitation of previous allele-specific extension assays in which slow reaction kinetics will still give rise to extension products from mismatched 3'-end primers, hindering proper discrimination. It thus represents a significant improvement of the allele-extension method. AMASE was evaluated by a bioluminometric assay in which successful incorporation of unmodified nucleotides is monitored in real-time using an enzymatic cascade.

Alleles↗

PubMed2001

Method enabling fast partial sequencing of cDNA clones.

Pyrosequencing is a nonelectrophoretic single-tube DNA sequencing method that takes advantage of cooperativity between four enzymes to monitor DNA synthesis. To investigate the feasibility of the recently developed technique for tag sequencing, 64 colonies of a selected cDNA library from human were sequenced by both pyrosequencing and Sanger DNA sequencing. To determine the needed length for finding a unique DNA sequence, 100 sequence tags from human were retrieved from the database and different lengths from each sequence were randomly analyzed. An homology search based on 20 and 30 nucleotides produced 97 and 98% unique hits, respectively. An homology search based on 100 nucleotides could identify all searched genes. Pyrosequencing was employed to produce sequence data for 30 nucleotides. A similar search using BLAST revealed 16 different genes. Forty-six percent of the sequences shared homology with one gene at different positions. Two of the 64 clones had unique sequences. The search results from pyrosequencing were in 100% agreement with conventional DNA sequencing methods. The possibility of using a fully automated pyrosequencer machine for future high-throughput tag sequencing is discussed.

Base Sequence↗

PubMed2001

Typing of human papillomavirus by pyrosequencing.

The possibility of using a new bioluminometric DNA sequencing technique, called pyrosequencing, for typing of human papillomaviruses (HPV) was investigated. A blinded pyrosequencing test was performed on an HPV test panel of 67 GP5+/GP6+ PCR-derived amplification products. The 67 clinical DNA samples were sequenced up to 25 bases and sequences were searched using BLAST. All of the samples were correctly genotyped by pyrosequencing and the results were unequivocally in accordance with the results obtained from conventional DNA sequencing. Pyrosequencing was found to be a fast and efficient tool for identifying individual HPV types. Furthermore, pyrosequencing has the capability of determining novel HPV types as well as HPV sequence variants harboring mutation(s). The method is robust and well suited for large-scale programs.

Base Sequence↗

PubMed2001

Alteration of interleukin 2 (IL-2) pharmacokinetics and function by IL-2 antibodies induced after treatment of colorectal carcinoma patients with a combination of monoclonal antibody 17-1A, granulocyte macrophage colony-stimulating factor, and IL-2.

In this study, we have assessed the development of neutralizing and non-neutralizing interleukin 2 (IL-2) antibodies in metastatic colorectal carcinoma patients receiving a colon carcinoma reactive monoclonal antibody (17-1A) in combination with granulocyte macrophage colony-stimulating factor and IL-2 therapy. Before treatment, no IL-2 antibodies were detected in any of the patients. After therapy, 10 of the 19 patients tested developed antibodies that bound to the IL-2 product used for therapy, but only one developed antibodies that neutralized the biological activity of IL-2 as assessed using an in vitro bioassay. We found that the induction of IL-2 antibodies in some patients irrespective of their neutralizing potential had a significant impact on IL-2 pharmacokinetics. A significant reduction of the area under the concentration-time curve and maximum concentration (C(max)) and increased IL-2 distribution and clearance were observed in IL-2 antibody-positive patients in comparison with IL-2 antibody-negative patients. A significant decrease in IL-2-mediated expansion of lymphocytes was also evident in patients positive for IL-2 antibodies in comparison with those negative for these antibodies. Further characterization of sera from patients with antibodies showed that, in most cases, the antibodies recognized different IL-2 preparations. Results also showed that serum IL-2 concentration at initiation of therapy in patients was significantly higher relative to healthy control donors. The endogenous production of IL-2 gradually increased during the treatment cycles. To conclude, induction of neutralizing and non-neutralizing antibodies in cytokine-treated patients should be carefully monitored in terms of their clinical significance.

Adult↗

PubMed2000

Mutation detection by pyrosequencing: sequencing of exons 5-8 of the p53 tumor suppressor gene.

The ability to sequence a large number of DNA samples rapidly and accurately for detection of all possible mutations is a critical goal for the future application of DNA sequencing in routine medical diagnostics. Pyrosequencing() is a non-electrophoretic real-time DNA sequencing method that uses the luciferase-luciferin light release as the detection signal for nucleotide incorporation into target DNA. For pyrosequencing of the human p53 gene, a nested multiplex PCR method for amplification of exons 5-8 was prepared. In order to investigate the use of pyrosequencing in mutation detection, DNA samples from skin-cancer patients were used. Two forms of nucleotide dispensation strategy were used, cyclic and programmed. Bi-directional pyrosequencing was performed and the overlapping sequence data produced were assembled to determine the sequence of the gene. Reliable sequencing data were obtained with both dispensation strategies, but some advantages were obtained using the programmed nucleotide dispensation approach, such as longer and faster reads, and fewer out-of-phase problems. The accuracy of pyrosequencing for detection of p53 mutations and allele distribution was demonstrated.

Base Sequence↗

PubMed2000

Single-nucleotide polymorphism analysis by pyrosequencing.

There is a growing demand for high-throughput methods for analysis of single-nucleotide polymorphic (SNP) positions. Here, we have evaluated a novel sequencing approach, pyrosequencing, for such purposes. Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzymatic reactions yields detectable light, which is proportional to incorporated nucleotides. One feature of typing SNPs with pyrosequencing is that each allelic variant will give a unique sequence compared to the two other variants. These variants can easily be distinguished by a pattern recognition software. The software displays the allelic alternatives and allows for direct comparison with the pyrosequencing raw data. For optimal determination of SNPs, various protocols of nucleotide dispensing order were investigated. Here, we demonstrate that typing of SNPs can efficiently be performed by pyrosequencing using an automated system for parallel analysis of 96 samples in approximately 5 min, suitable for large-scale screening and typing of SNPs.

Base Sequence↗