Biomedical subjects

E Gentalen

Publications and source records attributed to E Gentalen.

3 recordsLinked to original sources

PubMed1999

Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

Culture Media↗

PubMed1999

A novel method for determining linkage between DNA sequences: hybridization to paired probe arrays.

Cooperative hybridization has been used to establish physical linkage between two loci on a DNA strand. Linkage was detected by hybridization to a new type of high-density oligonucleotide array. Each synthesis location on the array contains a mixture of two different probe sequences. Each of the two probes can hybridize independently to a different target sequence, but if the two target sequences are physically linked there is a cooperative increase in hybridization yield. The ability to create and control non-linear effects raises a host of possibilities for applications of oligonucleotide array hybridization. The method has been used to assign linkage in 50:50 mixtures of DNA containing single nucleotide polymorphisms (SNPs) separated by 17, 693, 1350 and 2038 bp and to reconstruct haplotypes. Other potential uses include increasing the specificity of hybridization in mutation detection and gene expression monitoring applications, determining SNP haplotypes, characterizing repetitive sequences, such as short tandem repeats, and aiding contig assembly in sequen-cing by hybridization.

Genetic Linkage↗

PubMed1999

Quantifying DNA-protein interactions by double-stranded DNA arrays.

We have created double-stranded oligonucleotide arrays to perform highly parallel investigations of DNA-protein interactions. Arrays of single-stranded DNA oligonucleotides, synthesized by a combination of photolithography and solid-state chemistry, have been used for a variety of applications, including large-scale mRNA expression monitoring, genotyping, and sequence-variation analysis. We converted a single-stranded to a double-stranded array by synthesizing a constant sequence at every position on an array and then annealing and enzymatically extending a complementary primer. The efficiency of second-strand synthesis was demonstrated by incorporation of fluorescently labeled dNTPs (2'-deoxyribonucleoside 5'-triphosphates) and by terminal transferase addition of a fluorescently labeled ddNTP. The accuracy of second-strand synthesis was demonstrated by digestion of the arrayed double-stranded DNA (dsDNA) on the array with sequence-specific restriction enzymes. We showed dam methylation of dsDNA arrays by digestion with DpnI, which cleaves when its recognition site is methylated. This digestion demonstrated that the dsDNA arrays can be further biochemically modified and that the DNA is accessible for interaction with DNA-binding proteins. This dsDNA array approach could be extended to explore the spectrum of sequence-specific protein binding sites in genomes.

Base Sequence↗