Comparative gene expression profiling by oligonucleotide fingerprinting

Meier‐Ewert, Sebastian

doi:10.1093/nar/26.9.2216

Cited by 64 publications

(50 citation statements)

References 20 publications

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“…PCR reactions are then carried out with these primers on a template DNA. In the next step, an oligofingerprinting (OFP) process [22][23][24][25][26] is used to characterize clones by their patterns of hybridization with a series of up to 200 short oligonucleotides. The pattern of hybridization for each clone is called its fingerprint.…”

Section: Resultsmentioning

confidence: 99%

“…We generated robotically high-density filter arrays of the PCR-amplified OST clones as described [24]. All OST clones were spotted as quadruplets onto 22 cm ϫ 22 cm nylon membranes.…”

Section: Methodsmentioning

confidence: 99%

“…The primers were used in a series of PCR reactions on human genomic DNA [7]. We applied to the library of PCR products, consisting of 13,580 clones, an oligonucleotide fingerprinting procedure with a set of 193 8-mer oligonucleotide probes [22][23][24][25][26]. Cluster analysis of the clone fingerprints [27] revealed 239 clusters and 121 singletons (single clone clusters).…”

Section: Deciphering the Human Or Gene Superfamilymentioning

confidence: 99%

“…To overcome such redundancy, we applied oligonucleotide fingerprinting (OFP). This technique was previously used successfully on cDNA and shotgun libraries [22][23][24][25][26]. In OFP, each clone of the library obtains a unique fingerprint of its hybridization pattern with a set of short oligonucleotides.…”

Section: Figmentioning

confidence: 99%

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DEFOG: A Practical Scheme for Deciphering Families of Genes

et al. 2002

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We developed a novel efficient scheme, DEFOG (for "deciphering families of genes"), for determining sequences of numerous genes from a family of interest. The scheme provides a powerful means to obtain a gene family composition in species for which high-throughput genomic sequencing data are not available. DEFOG uses two key procedures. The first is a novel algorithm for designing highly degenerate primers based on a set of known genes from the family of interest. These primers are used in PCR reactions to amplify the members of the gene family. The second combines oligofingerprinting of the cloned PCR products with clustering of the clones based on their fingerprints. By selecting members from each cluster, a low-redundancy clone subset is chosen for sequencing. We applied the scheme to the human olfactory receptor (OR) genes. OR genes constitute the largest gene superfamily in the human genome, as well as in the genomes of other vertebrate species. DEFOG almost tripled the size of the initial repertoire of human ORs in a single experiment, and only 7% of the PCR clones had to be sequenced. Extremely high degeneracies, reaching over a billion combinations of distinct PCR primer pairs, proved to be very effective and yielded only 0.4% nonspecific products. characterized in rat a decade ago [2] and have since been detected in many vertebrate species [reviewed in 6]. So far, about 3000 OR genes and pseudogenes are known in 24 vertebrate species [7][8][9][10][11][12]. They are divided into 32 distinct families based on phylogenetic analysis [8].Roughly 900 OR coding sequences were found in the first draft of the human genome [9,10]. As predicted [7,13], between 53% and 63% of them have frame disruptions and are therefore considered as pseudogenes. OR genes are found on almost all human chromosomes except chromosome 20 and Y [7,9,10,13,14]. Almost 80% of the ORs are organized in clusters of six or more genes [9,10]. This is in good agreement with previous fluorescence in situ hybridization (FISH) experiments and sequencing data [7,13,14]. 296Article doi:10.1006/geno.2002.6830, available online at http://www.idealibrary.com on IDEAL The coding region of OR genes spans approximately 1 kb. This region lacks introns [2,13] and is preceded by a large intron and several short noncoding exons [16][17][18][19][20]. Inside the coding region there are several conserved segments [21] that allow easy amplification of the intronless coding region from genomic DNA by PCR assay. The PCR product is termed the "olfactory receptor sequence tag" (OST) [7].We developed and experimentally tested a practical scheme for deciphering families of genes (DEFOG). The scheme provides a powerful means to obtain a sequence composition of a gene family in species for which high-throughput genomic sequencing data are unavailable. To validate DEFOG, we tested it on the human OR gene superfamily. Starting with a limited number of human ORs, it almost tripled the size of this set in a single experiment. We suggest that DEFOG can be successfully appli...

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Section: Resultsmentioning

confidence: 99%

“…We generated robotically high-density filter arrays of the PCR-amplified OST clones as described [24]. All OST clones were spotted as quadruplets onto 22 cm ϫ 22 cm nylon membranes.…”

Section: Methodsmentioning

confidence: 99%

Section: Deciphering the Human Or Gene Superfamilymentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

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DEFOG: A Practical Scheme for Deciphering Families of Genes

et al. 2002

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“…A re-arrayed oligo-fingerprinted shield stage library (RZPD library number 637) was amplified by PCR in 384-well plates by using M13FSP and 3/86 primers as described previously (Meier-Ewert et al, 1998;Clark et al, 1999Clark et al, , 2001) and spotted on 22 ϫ 22 cm Hybondϩ nylon filters (Amersham, UK), by using an in-house built and designed spotting robot. Each cDNA clone was spotted in duplicate, to allow quality control of hybridization results.…”

Section: Generation Of Gastrula Stage Arraysmentioning

confidence: 99%

Identification of nodal signaling targets by array analysis of induced complex probes

Dickmeis

Aanstad

Clark

et al. 2001

Developmental Dynamics

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Nodal signaling controls germ layer formation, left-right asymmetry, and patterning of the brain in the vertebrate embryo. Cellular responses to Nodal signals are complex and include changes in gene expression, cell morphology, and migratory behavior. Only little is known about the genes regulated by Nodal signaling. We designed a subtractive screening strategy by using a constitutively active Nodal receptor to identify putative target genes of Nodal signals in the early gastrula of zebrafish embryos. By quantitative analysis of macro-array hybridizations, 132 genes corresponding to 1.4% of genes on the entire macro-array were identified, which were enriched in the Nodal-induced probe pool. These genes encode components of signal transduction pathways, transcription regulators, proteins involved in protein metabolism but also cytoskeletal components and metabolic enzymes, suggesting dramatic changes of cell physiology in gastrula cells in response to Nodal signals.

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Gene Expression Analysis

Speed

2005

Encyclopedia of Biostatistics

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Comparative gene expression profiling by oligonucleotide fingerprinting

Cited by 64 publications

References 20 publications

DEFOG: A Practical Scheme for Deciphering Families of Genes

DEFOG: A Practical Scheme for Deciphering Families of Genes

Identification of nodal signaling targets by array analysis of induced complex probes

Gene Expression Analysis

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