Biomedical subjects

B Darnhofer-Demar

Publications and source records attributed to B Darnhofer-Demar.

4 recordsLinked to original sources

PubMed1998

Sequencing exons 5 to 8 of the p53 gene by MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption ionization time of flight mass spectrometry was used to sequence exons 5 to 8 of the human p53 gene. A single tube procedure was established for target amplification and mass spectrometric (MS) sequencing. The MS sequencing scheme is designed for high throughput and parallel sample processing, and is amenable to full automation. Reliable sequencing data were obtained using fmol sample amounts. The high resolution and accuracy of MS sequencing was demonstrated by direct sequencing of a heterozygous template.

Base Sequence↗

PubMed1997

Detection of RET proto-oncogene codon 634 mutations using mass spectrometry.

Mutations located in the RET proto-oncogene at codon 634 associated with multiple endocrine neoplasia type 2A and medullary thyroid carcinoma are detected by low-resolution and high-resolution mass spectrometry schemes not requiring labeling or electrophoretic separation of diagnostic products. The former requires measurement by matrix-assisted laser desorption ionization time-of-flight mass spectrometry of 21- to 27-mer oligonucleotides generated by a primer oligo base extension reaction. The latter is based upon direct measurement of artificial products which include the mutation site using matrix-assisted laser desorption ionization Fourier transform mass spectrometry. In this feasibility study a synthetic 25-mer representing the wildtype allele (7660.3 Da) was easily distinguished from G to A (7644.3 Da) and G to T (7635.3 Da) mutant alleles; the mutant alleles, which differed in mass by only 9.0 Da, were easily resolved when analyzed as a mixture. The results of both detection schemes were highly accurate and reliable, indicating mass spectrometry to be a high-quality alternative for future DNA diagnostics performed in clinical laboratories and genetic profiling studies.

Base Sequence↗

PubMed1997

Identification of apolipoprotein E polymorphisms using temperature cycled primer oligo base extension and mass spectrometry.

The isothermal Primer Oligo Base Extension (PROBE) reaction combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for diagnostic product detection as recently introduced by our group is modified to incorporate temperature cycling during the primer extension step, resulting in enhanced levels of diagnostic product generation. Utilizing temperature cycled PROBE, the identities of two apolipoprotein E polymorphisms (codons 112 and 158) for differentiation of epsilon 2/epsilon 3, epsilon 3/epsilon 3, epsilon 3/epsilon 4, and epsilon 4/epsilon 4 genotypes were simultaneously determined. Primers specific for each site are extended by a series of bases unique to the identity of that variable site, producing low mass diagnostic products (M(r) < 9000) highly amenable to detection by mass spectrometry. The temperature cycled PROBE method has yielded unambiguous and correct diagnoses for all samples tested thus far. The increased amount of diagnostic product generated per primer by the cycling method makes possible faster spectrum acquisition due to the increased signal intensity, critical for future automated measurement of such samples.

Apolipoproteins E↗

PubMed1995

Rapid purification of a functionally active plant sucrose carrier from transgenic yeast using a bacterial biotin acceptor domain.

A rapid and efficient method has been used for the purification of a Plantago major sucrose carrier from Saccharomyces cerevisiae. The C-terminal fusion of a bacterial biotin acceptor domain to the carrier protein did not interfere with the targeting to the yeast plasma membrane nor with the catalytic activity of the sucrose carrier. The chimeric construct is biotinylated by yeast cells in vivo and represents the only biotinylated protein in yeast membranes. Solubilized biotinylated carrier protein binds selectively to immobilized monomeric avidin and can be eluted as pure protein with free biotin. The purified protein is functionally active and catalyzes the energy-dependent transport of sucrose into proteoliposomes.

Amino Acid Sequence↗