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D H Farkas

Publications and source records attributed to D H Farkas.

17 recordsLinked to original sources
PubMed2000

Bioelectronic detection of point mutations using discrimination of the H63D polymorphism of the Hfe gene as a model.

BACKGROUND: A bioelectronic detection platform has recently been developed that facilitates the detection and characterization of nucleic acids. The DNA chip platform is compatible with homogeneous assays because separate labeling and wash steps are not required. A one-step, bioelectronic detection assay was developed to genotype patient samples with respect to the H63D polymorphism of the Hfe gene, associated with hereditary hemochromatosis. METHODS AND RESULTS: Electrode arrays were modified with DNA capture probes that were perfectly matched to the wild-type or mutant allele of H63D. Amplicons containing the polymorphic site were hybridized with the capture probes on the electrode arrays in the presence of electronically labeled reporter (signaling) probes. Voltammetric analysis of the electrode arrays was conducted first at ambient temperature and then at elevated temperature. The electronic signal was preferentially diminished at elevated temperature from electrodes that hybridized with mismatched target amplicons. CONCLUSION: An assay for bioelectronic genotyping of the H63D polymorphism was developed and used with six patient specimens to show the feasibility of this system as a model for point mutation detection.

Amino Acid Substitution↗
PubMed1996

Specimen collection and storage for diagnostic molecular pathology investigation.

The success of the newest discipline in the diagnostic clinical pathology laboratory, molecular pathology, is dependent on proper collection and storage of both the original and processed (nucleic acid) specimen. This issue will grow in importance as test volumes increase in the diagnostic molecular pathology laboratory. This review is a distillation of a literature review by the Patient Preparation and Specimen Handling Committee of the College of American Pathologists. It describes specific collection, storage, and anticoagulant or preservative requirements based on the diagnostic molecular technique and/or specimen type used for analysis. This review serves as a guide for clinical laboratories interested in appropriate collection and storage of specimens to be used in nucleic acid-based analysis.

DNA↗
PubMed1996

Mitochondrial disorders. Methods and specimen selection for diagnostic molecular pathology.

Mitochondrial DNA is a circular double-stranded macromolecule. Each strand contains 16 569 base pairs. Mutations in mitochondrial DNA, including base substitutions in tRNA or rRNA genes, deletions, duplications, or base substitutions in genes for protein subunits, lead to specific diseases. The ratio of mutated to normal mitochondrial DNA may vary from tissue to tissue (heteroplasmy) in mitochondrial DNA diseases. Therefore, the source of the specimen is important in the evaluation of mitochondrial DNA mutations. Detection method selection is also critical. For example, single-strand conformation polymorphism is not as specific for tRNA mutations as is gene sequencing or amplification of a specific gene by polymerase chain reaction. Care in both specimen collection and analytic method are important in the successful evaluation of patients with a potential mitochondrial DNA disease.

DNA, Mitochondrial↗
PubMed1996

Specimen stability for DNA-based diagnostic testing.

The use of molecular diagnostic testing is increasing in the clinical setting; therefore, data regarding DNA stability in clinical specimens are essential for correct test performance and interpretation. This study was designed to determine DNA stability in peripheral blood and solid tissue under different storage conditions. DNA quality and yield were assayed by spectrophotometric absorbance, gel electrophoresis, and suitability for Southern hybridization and polymerase chain reaction (PCR), the most widely employed clinical DNA analyses. A second goal of the study was to evaluate DNA stability during storage at 4 degrees C for 1 month to 3 years. The data show that freezing or refrigeration of separated leukocytes is preferable for short- to intermediate-term storage and freezing is preferable for solid tissue. DNA degradation varying from slight to severe is seen inconsistently with such specimens, probably due to sampling of unevenly frozen-tissue areas. Depending on the degree of DNA degradation, analysis may still be possible by PCR and in some cases even by Southern hybridization. Once isolated, DNA was stable at 4 degrees C for at least 3 years. These results suggest a more flexible approach to specimen requirements for molecular pathology, as some samples that would routinely be rejected gave interpretable results.

Blood Cells↗
PubMed1993

Improved quantitation of HER-2/neu gene copy number in breast tumor-derived DNA samples.

A Southern blot-based assay is presented that increases the simplicity and accuracy of the HER-2/neu gene copy number assignment in breast cancer DNA. Genomic DNA was hybridized simultaneously with probes corresponding to portions of HER-2/neu and a single-copy gene, myeloperoxidase. A unique restriction fragment was detected for each gene. The use of DNA probes of similar mass and equal specific activities resulted in a ratio of band intensities on the resultant autoradiograph that reported the ratio of gene copy numbers directly. Patient samples containing amplified levels of the HER-2/neu gene were identified by simple visual inspection of a single autoradiograph. Analysis of breast cancer samples alongside the cell line DNAs, representing a range of HER-2/neu gene copy numbers, permits visual quantitation of the tumors' gene copy numbers. The authors show that the HER-2/neu gene copy number can be determined accurately in marginally degraded DNA, a feature of some clinical samples.

Autoradiography↗
PubMed1991

Inhibition of transient gene expression with plasmids encoding herpes simplex virus type 1 UL55 and alpha genes.

Herpes simplex virus type 1 (HSV-1) subgenomic sequences from 0.743 to 0.782 map units have been molecularly cloned as plasmid AT1 and shown to inhibit stable DNA-mediated gene transformation of Ltk- cells with the HSV-1 thymidine kinase (tk) gene. Here it is shown that AT1 also inhibits transient gene expression. Expression from the chloramphenicol acetyltransferase (CAT) gene under the control of either the HSV-1 tk gene or the Rous sarcoma virus (RSV) promoter was inhibited when cotransfected into Ltk- and CV-1 cells with equimolar amounts of AT1. AT1 was subcloned as three overlapping plasmids called AT1a, alpha 27 and AT1b. The alpha 27 plasmid encodes the HSV-1 immediate early gene, alpha 27; AT1a possesses sequences that specify an open reading frame in HSV-1 strain KOS used in these studies, although the significance of this open reading frame is unknown; AT1b possesses the sequences for UL55 and UL56, also genes for which no function has been reported. No single subclone or pair of subclones demonstrated significant inhibition of transient gene expression. Cotransfection of all three subclones did result in inhibition of RSV-CAT gene expression, suggesting that information from each subclone is necessary. One of the three subclones, alpha 27, contains the HSV-1 immediate early gene, alpha 27, so the possibility that other immediate early genes could substitute for alpha 27 was tested. Inhibition of RSV-CAT gene expression was also achieved by cotransfection of AT1a and AT1b with either an alpha 0- or alpha 4-containing plasmid, suggesting that the role of the alpha 27-containing plasmid can be replaced by other alpha genes with trans-regulating capability. Finally, AT1a and AT1b linker insertion mutants have been constructed and used to study the role these plasmids play in mediating inhibition. These results suggest that AT1 contains HSV-1 functions in addition to that of alpha 27 that interfere with gene expression.

Animals↗
PubMed1987

In situ staining procedure for the detection of ornithine transcarbamylase activity in polyacrylamide gels.

Ornithine transcarbamylase deficiency is a human genetic disease potentially susceptible to gene therapy. A murine model system exists for the disease in the sparse-fur (spf) mouse. Before gene therapy studies can be performed it is necessary to have practical methods which could detect successful gene transfer. Therefore we have developed an in situ staining procedure for the detection of ornithine transcarbamylase activity in polyacrylamide gels. Following electrophoretic separation under nondenaturing conditions inorganic phosphate cleaved from carbamyl phosphate in gels as a result of enzymatic activity was precipitated as phosphomolybdic acid and visualized by reduction with ascorbic acid. Results from the procedure correlated with ornithine transcarbamylase activity as measured by solution assay for citrulline, the other product of the reaction. This procedure readily distinguished mutant forms of ornithine transcarbamylase as exemplified by the murine spf mutation and resolved ornithine transcarbamylases of all animals tested into multiple forms. The procedure further distinguished ornithine transcarbamylases of animals of several different genera while yielding virtually identical patterns of the enzyme from species within the same genus. This procedure also suggested that the human enzyme was more labile than murine ornithine transcarbamylase; direct thermolability studies confirmed this finding.

Animals↗
PubMed1987

Sequences of herpes simplex virus type 1 that inhibit formation of stable TK+ transformants.

We have identified two regions of the herpes simplex virus type 1 (HSV-1) genome that inhibit DNA-mediated transformation of thymidine kinase-less L (Ltk-) cells by the cloned HSV-1 tk gene. When plasmids containing the EcoRI fragments EK or JK were mixed at 30 fmol/ml with the tk gene and transfected into Ltk- cells, the frequency of transformation was inhibited 80 to more than 90% relative to the control. Of the remaining 10 EcoRI fragments of the HSV-1 genome, 8 were inactive and 2 were weakly active. A 6.1-kilobase PstI subclone between 0.743 and 0.782 map units was isolated from pEK. This clone, pEK-P3P4, exhibited antitransformation activity toward HSV-1 tk and also the bacterial genes gpt and neo. pEK-P3P4 contains the alpha 27 gene, and restriction endonuclease inactivation and subcloning studies established that alpha 27 alone did not inhibit transformation. However, alpha 27 plus sequences both upstream and downstream of alpha 27 did inhibit transformation. In addition, alpha 0 or alpha 4 could substitute for alpha 27 in effecting antitransformation with these sequences. Therefore, an alpha gene and two additional loci in pEK-P3P4 are required for antitransformation. A second antitransforming locus in the reiterated sequences common to EK and JK and distinct from those in pEK-P3P4 was also identified but not characterized in detail. How antitransformation may be an expression of regulation of viral and host cell gene expression is discussed.

Base Sequence↗
PubMedDate not supplied

B- and T-cell gene rearrangement test: an overview of application and methodology.

OBJECTIVE: To examine the use of the B- and T-cell rearrangement test in the diagnosis of lymphoid neoplasms. DATA SOURCES: Recent texts, professional journals, and authors' experiences. STUDY SELECTION: Not specified. DATA EXTRACTION: Not specified. DATA SYNTHESIS: The B- and T-cell rearrangement test can detect a monoclonal population of B- and T-cells, strongly indicative of neoplasia. This is accomplished through the use of DNA probes. The presence of a unique band on the Southern blot signifies a monoclonal gene rearrangement, which can make or confirm a diagnosis of a lymphoproliferative disorder and classify the lineage as B- or T-cell. T-cell neoplasms generally behave more aggressively than B-cell neoplasms, which can have important implications for prognosis and therapy. The unique gene rearrangement "signature" can be followed during and after therapy to document remission or recurrence. The most commonly examined samples are from the blood, bone marrow, and lymph nodes, but any tissue or fluid suspected of harboring a lymphoid neoplasm can be examined. CONCLUSION: The test is an important diagnostic indicator in the evaluation and follow-up of patients with lymphoma or lymphoid leukemia. It is most helpful when it can be correlated to the clinical condition of the patient and other ancillary studies.

Blotting, Southern↗
PubMedDate not supplied

Immunohistochemical analysis of P-glycoprotein expression in breast cancer: clinical correlations.

Paraffin-embedded, formalin-fixed tissue (PEFFT) specimens from a subset of breast cancer patients were analyzed by immunohistochemistry to determine whether or not the presence of P-glycoprotein identified chemotherapy resistance. Two antibodies, C219 (monoclonal) and Ab1 (polyclonal), demonstrated appropriate immunostaining. Retrospectively and prospectively, P-glycoprotein expression was determined from PEFFT of 20 breast cancer biopsies (19 patients with locally advanced or metastatic disease). Immunohistochemical staining was graded for number of positive cells (N0 to N4) and intensity (I0 to I3). The immunostaining N and I of both antibodies were similar. There was no correlation between N, (P = 0.13) or I, (P = 0.67) and chemotherapy response or between N, (P = 0.63) or I, (P = 0.89) and survival. Five patients had residual cancer at repeat biopsy after systemic chemotherapy for locally advanced disease. These specimens had similar N and I as the primary cancer. This assay accurately identifies P-glycoprotein expression in PEFFT and revealed no correlation between expression and chemotherapy response or survival.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMedDate not supplied

Bone marrow biopsy imprint preparations: use for molecular diagnostics in leukemias.

Bone marrow (BM) biopsies occasionally fail to yield aspirate specimens; such "dry taps" pose diagnostic difficulties. In the absence of a BM aspirate, morphological evaluation and cytochemistry rely on core biopsy imprint preparations (IP) and other analyses, e.g., molecular pathology studies, become impossible. The use of imprint preparations (IPs) for molecular diagnostics based on messenger ribonucleic acid (mRNA) analysis by reverse transcription-polymerase chain reaction (RT-PCR) for detection of breakpoint cluster region (bcr) gene rearrangements in chronic myelogenous leukemia (CML) and myeloperoxidase (MPO) mRNA in acute leukemias (AL) is described. Fifteen IPs from five core biopsies (three CML and two AL) were used. Analysis of bcr was positive in all nine IPs from the three cases of CML and was confirmed on peripheral blood samples. Detection of MPO mRNA established the myeloid lineage of the blasts in two cases of AL, when cytochemistry and flow cytometry failed to reveal myeloid markers. These tests are useful when fresh BM aspirate is unobtainable, since multiple imprints of the biopsy are easily prepared at the bedside.

Biopsy↗