Biomedical subjects

D Siemieniak

Publications and source records attributed to D Siemieniak.

7 recordsLinked to original sources

PubMed1998

Genotyping of apolipoprotein E by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The genotyping of the various isoforms of Apolipoprotein E (apo E) has been performed using matrix-assisted laser desorption/ionization (MALDI-MS). The polymerase chain reaction was used to amplify the specific apo E gene sequence followed by digestion with Cfo I (Clostridium formicoaceticum), for generating restriction fragments for rapid and accurate mass analysis. An exonuclease I digestion step was introduced to remove the unused primers after PCR, which can otherwise interfere in the mass spectral analysis. By replacing the gel electrophoresis detection step with MALDI-MS, restriction isotyping of the apo E gene was achieved. Genotyping of an unknown sample and obtained from an independent diagnostic laboratory demonstrated the validity of the MALDI-MS method for the routine analysis of apo E.

Apolipoproteins E↗

PubMed1997

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as a rapid screening method to detect mutations causing Tay-Sachs disease.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used as a rapid method for the detection of human genetic polymorphisms. In particular, the mutations in the human HEXA gene that cause the infantile Tay-Sachs disease have been studied using MALDI-MS to demonstrate the feasibility of this technique for use in clinical and diagnostic analysis. The protocols involved in this approach include, polymerase chain reaction for the amplification of the mutation site from buccal cell DNA, followed by restriction enzyme digestion of the amplified regions of the template cells. The products of amplification and digestion were studied using MALDI-MS. MALDI-MS experiments are shown to provide essentially the same information as obtained from gel electrophoresis but orders of magnitude faster.

Base Sequence↗

PubMed1995

Rapid screening of genetic polymorphisms using buccal cell DNA with detection by matrix-assisted laser desorption/ionization mass spectrometry.

A new approach is developed for the rapid and cost-effective detection of human genetic polymorphisms based on matrix-assisted laser description/ionization mass spectrometric (MALDI MS) detection using a nitrocellulose film substrate. This method employs polymerase chain reaction (PCR) amplification using DNA extracted from buccal cells as templates, followed by direct digestion with restriction enzymes and subsequent analysis by MALDI MS. The extraction of DNA from buccal cells provides a rapid and convenient means for sampling PCR-based diagnostic analysis. The amount of DNA was sufficient as the template for both normal PCR amplifications, and amplifications involving the use of mismatched primers and multiple primers. The MALDI MS methodology has been successfully used for the analysis of such PCR products where restriction fragments generated directly in PCR reactions have been used for detection of carbonic anhydrase and cystic fibrosis transmembrane conductance regulator as model genes. The detection of genetic polymorphisms following routine biological and clinical procedures with the MALDI MS method is demonstrated. The results from MALDI MS analysis are shown to be comparable to those obtained from gel electrophoresis but the MALDI MS method is several orders of magnitude faster than gel electrophoretic techniques. The method described herein should also be readily extended to other areas involving DNA screening and testing.

Base Sequence↗

PubMed1994

Matrix-assisted laser desorption/ionization mass spectrometry of restriction enzyme-digested plasmid DNA using an active Nafion substrate.

Matrix-assisted laser desorption/ionization using a 3-hydroxypicolinic acid matrix from an active Nafion substrate has been used for detection of restriction enzyme-digested double-stranded plasmid DNA using time-of-flight mass spectrometry. DNA strands of up to 267 base pairs were detected with minimal sample purification, although only as species corresponding to single-stranded DNA.

Base Sequence↗

PubMed1993

Isolation of a partial candidate gene for Menkes disease by positional cloning.

Menkes disease is an X-linked recessive disorder of copper metabolism resulting in death in early infancy. The gene has been mapped to band Xq13 based, in part, on a translocation breakpoint in a female with the disease, which was found to lie within 300 kilobases (kb) of the PGK-1 locus, allowing the isolation of a YAC clone spanning the breakpoint. Phage subclones from the breakpoint region were isolated and used to screen cDNA libraries. cDNA clones were found which detect an 8 kb transcript from normal individuals but show diminished or absent hybridization in Menkes disease patients. Partial sequence of the cDNA shows a unique open reading frame containing putative metal binding motifs which have been found in heavy metal resistance genes in bacteria. This gene is a strong candidate for the Menkes disease gene.

Adenosine Triphosphatases↗

PubMed1989

Tarsius delta- and beta-globin genes: conversions, evolution, and systematic implications.

Comparisons between duplicated genes have shown that gene conversions play an important role in the evolution of multigene families. Previous comparisons have documented in the recently duplicated gamma-fetal globin genes of catarrhine primates, over 15 separate conversions affecting extensive stretches of coding and noncoding sequences. In the present study, delta- and beta- globin genes from a lower primate Tarsius syrichta, and the delta-globin gene of the Asian great ape, Pongo pygmaeus, have been isolated and sequenced. Comparisons of these sequences with other primate delta and beta sequences confirmed a previously reported conversion in an anthropoid ancestor and revealed additional conversions in basal primate, stem haplorhine, tarsier, and early lemur lineages. Conversions found between primate delta- and beta-globin genes contrast with those found in the gamma-genes in that delta-beta conversions appear much less frequently and are more restricted to regions conserved by selection (i.e. coding and 5'-regulatory sequences). These differences indicate that soon after a duplication occurs, conversions can be quite frequent and encompass extensive portions of the duplicated region. With time, sequence differences accumulate, particularly in noncoding regions, and limit both the frequency and size of the conversions. Sequences conserved by selection accumulate differences more slowly and are therefore subject to gene conversions for a longer period of time. Both unconverted and converted sequences were consistent in supporting the placement of tarsier with anthropoids.

Animals↗

PubMed1988

Nucleotide sequence of a mutation in the proB gene of Escherichia coli that confers proline overproduction and enhanced tolerance to osmotic stress.

We determined the nucleotide (nt) sequence of a mutation that confers proline overproduction and enhanced tolerance of osmotic stress on bacteria. The mutation, designated as proB74, is an allele of the Escherichia coli proB gene which results in a loss of allosteric regulation of the protein product, gamma-glutamyl kinase. Our sequencing indicated that the proB74 mutation is a substitution of an A for a G at nt position 319 of the coding strand of the gene, resulting in a change of an aspartate to an asparagine at amino acid (aa) residue 107 of the predicted protein product. Rushlow et al. [Gene 39 (1984) 109-112] determined that another proB mutation (designated as DHPR), that resulted in a loss of allosteric inhibition by proline of the E. coli gamma-glutamyl kinase, was due to a substitution of an alanine for a glutamate at aa residue 143. Therefore, even though both the DHPR and the proB74 mutations caused a loss of allosteric inhibition of gamma-glutamyl kinase, they are due to different amino acid substitutions.

Alleles↗