Biomedical subjects

Saul Surrey

Publications and source records attributed to Saul Surrey.

24 records · Page 2Linked to original sources

PubMed2002

Assembly of human hemoglobin (Hb) beta- and gamma-globin chains expressed in a cell-free system with alpha-globin chains to form Hb A and Hb F.

Rates of in vitro synthesis of radiolabeled gamma and beta chains made in a cell-free transcription/translation system were similar, but expressed globin chains were unstable. The addition of unlabeled beta or gamma chains at the start of chain synthesis generated radiolabeled beta(4) or gamma(2) and gamma(4) chains, respectively. If unlabeled alpha-globin chains were added at the start of chain synthesis, then approximately equal amounts of radiolabeled alphabeta or alphagamma bands were generated. If unlabeled Hb A or Hb F was added to reactions containing radiolabeled alphabeta or alphagamma prior to electrophoresis, then radiolabeled Hb A or Hb F tetramers, respectively, were generated. If alpha chains were added after synthesis of radiolabeled gamma chains made in the presence of unlabeled gamma chains, then little radiolabeled alphagamma formed. In contrast, if alpha chains were added after synthesis of radiolabeled beta chains made in the presence of unlabeled beta chains, then radiolabeled alpha(2)beta(2) formed. These findings suggest that beta and gamma chains associate with alpha chains during or soon after translation. This would prevent the formation of unstable monomers as well as stable gamma(2) dimers and suggests that alpha chains may bind to nascent non-alpha chains, acting as folding catalysts to promote functional tetrameric hemoglobin formation in vivo.

Cell-Free System↗

PubMed2002

Kinetics of heterogeneous hybridization on indium tin oxide surfaces with and without an applied potential.

The rate of hybridization of oligonucleotide target sequences to chemically immobilized oligonucleotide probes has been studied both with and without an electrical field. The probe size was 20-24 nucleotides (nt) while the target size ranged from 157 to 864 nt. In agreement with previous studies, complete hybridization under normal conditions required 10-30 hours, depending on target size. The kinetics were characterized by a characteristic lag time followed by an asymptotic rise to the final value. In contrast, with an applied electrical field, all but the largest target hybridized in about 10 min while the longest hybridized within 1 h. Deleterious electrode reactions were avoided by close spacing of the anode and cathode and application of very small voltages. Our results suggest that probes and targets orient flat on the surface. A model is suggested to explain the kinetics observed that involves a series of surface states between initial target arrival and final hybridized state. Our results show that the electric field accelerated hybrid capture of solution-phase targets by surface-bound probes. This approach may have implications for enhancing array-based hybrid capture for mutation detection, copy number determination and/or gene expression profiling.

DNA↗

PubMed2002

Pyrosequencing for detection of mutations in the connexin 26 (GJB2) and mitochondrial 12S RNA (MTRNR1) genes associated with hereditary hearing loss.

Hereditary hearing loss (HHL) is one of the most common congenital disorders and is highly heterogeneous. Mutations in the connexin 26 (CX26) gene (GJB2) account for about 20% of all cases of childhood deafness, and approach 50% in documented recessive cases of non-syndromic hearing loss. In addition, a single mitochondrial DNA mutation, mt1555A>G, in the 12S rRNA gene (MTRNR1), is associated with familial cases of progressive deafness. Effective screening of populations for HHL necessitates rapid assessment of several of these potential mutation sites. Pyrosequencing links a DNA synthesis protocol for determining sequence to an enzyme cascade that generates light whenever pyrophosphate is released during primer strand elongation. We assessed the ability of Pyrosequencing to detect common mutations causing HHL. Detection of the most common CX26 mutations in individuals of Caucasian (35delG), Ashkenazi (167delT), and Asian (235delC, V37I) descent was confirmed by Pyrosequencing. A total of 41 different mutations in the CX26 gene and the mitochondrial mt1555A>G mutation were confirmed. Genotyping of up to six different adjacent mutations was achieved, including simultaneous detection of 35delG and 167delT. Accurate and reproducible results were achieved taking advantage of assay flexibility and experimental conditions easily optimized for a high degree of standardization and cost-effectiveness. The standardized sample preparation steps, including target amplification by PCR and preparation of single-stranded template combined with automated sequence reaction and automated genotype scoring, positions this approach as a potentially high throughput platform for SNP/mutation genotyping in a clinical laboratory setting. .

Amino Acid Substitution↗

PubMed2002

Molecular diagnostics: hurdles for clinical implementation.

In the coming years, molecular diagnostics will continue to be of critical importance to public health worldwide. It will facilitate the detection and characterization of disease, as well as monitoring of the drug response, and will assist in the identification of genetic modifiers and disease susceptibility. A wide range of molecular-based tests is available to assess DNA variation and changes in gene expression. However, there are major hurdles to overcome before the implementation of these tests in clinical laboratories, such as which test to employ, the choice of technology and equipment, and issues such as cost-effectiveness, accuracy, reproducibility, personnel training, reimbursement by third-party payers and intellectual property. At present, PCR-based testing predominates; however, alternative technologies aimed at reducing genome complexity without PCR are anticipated to gain momentum in the coming years. Furthermore, development of integrated chip devices ("lab-on-a-chip") should allow point-of-care testing and facilitate genetic readouts from single cells and molecules. Together with proteomic-based testing, these advances will improve molecular diagnostic testing and will present additional challenges for implementing such testing in health care settings.

Animals↗

PubMed2002

Analysis of clinically relevant single-nucleotide polymorphisms by use of microelectronic array technology.

BACKGROUND: Microelectronic DNA chip devices represent an emerging technology for genotyping. We developed methods for detection of single-nucleotide polymorphisms (SNPs) in clinically relevant genes. METHODS: Primer pairs, with one containing a 5'-biotin group, were used to PCR-amplify the region encompassing the SNP to be interrogated. After denaturation, the biotinylated strand was electronically targeted to discrete sites on streptavidin-coated gel pads surfaces by use of a Nanogen Molecular Workstation. Allele-specific dye-labeled oligonucleotide reporters were used for detection of wild-type and variant sequences. Methods were developed for SNPs in genes, including factor VII, beta-globin, and the RET protooncogene. We genotyped 331 samples for five DNA variations in the factor VII gene, >600 samples from patients with beta-thalassemia, and 15 samples for mutations within the RET protooncogene. All samples were previously typed by various methods, including DNA sequence analysis, allele-specific PCR, and/or restriction enzyme digestion of PCR products. RESULTS: Analysis of amplified DNA required 4-6 h. After mismatched DNA was removed, signal-to-noise ratios were >5. More than 940 samples were typed with the microelectronic array platform, and results were totally concordant with results obtained previously by other genotyping methods. CONCLUSIONS: The described protocols detect SNPs of clinical interest with results comparable to those of other genotyping methods.

Factor VII↗

PubMedDate not supplied

Interpretation of fetal hemoglobin only on newborn screening for hemoglobinopathy.

Newborn screening for hemoglobinopathies rarely produces a fetal hemoglobin only result; it is most consistent with beta-thalassemia major, although other diagnoses are possible. The authors describe two unrelated African-American babies born in North Carolina whose newborn screening revealed fetal hemoglobin only. Both had a relatively benign clinical and hematologic picture. Molecular analyses indicated that both children are compound heterozygotes for beta-thalassemia and pancellular (deletional) hereditary persistence of fetal hemoglobin, a rare and apparently benign condition. Accurate interpretation of the fetal hemoglobin only result on newborn screening requires thorough evaluation, including family studies and molecular analysis.

Black or African American↗