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A biomedically enriched collection of 7000 human ORF clones.

Rolfs A, Hu Y, Ebert L, Hoffmann D, Zuo D, Ramachandran N, Raphael J, Kelley F, McCarron S, Jepson DA, Shen B, Baqui MM, Pearlberg J, Taycher E, DeLoughery C, Hoerlein A, Korn B, LaBaer J - PLoS ONE (2008)

Bottom Line: Second, clones were selected to represent the best available GenBank reference sequence.The target gene list was compared with 4000 human diseases and over 8500 biological and chemical MeSH classes in approximately 15 Million publications recorded in PubMed at the time of analysis.The outcome of this analysis revealed that relative to the genome and the MGC collection, this collection is enriched for the presence of genes with published associations with a wide range of diseases and biomedical terms without displaying a particular bias towards any single disease or concept.

View Article: PubMed Central - PubMed

Affiliation: Harvard Institute of Proteomics, Harvard Medical School, Cambridge, Massachusetts, USA.

ABSTRACT
We report the production and availability of over 7000 fully sequence verified plasmid ORF clones representing over 3400 unique human genes. These ORF clones were derived using the human MGC collection as template and were produced in two formats: with and without stop codons. Thus, this collection supports the production of either native protein or proteins with fusion tags added to either or both ends. The template clones used to generate this collection were enriched in three ways. First, gene redundancy was removed. Second, clones were selected to represent the best available GenBank reference sequence. Finally, a literature-based software tool was used to evaluate the list of target genes to ensure that it broadly reflected biomedical research interests. The target gene list was compared with 4000 human diseases and over 8500 biological and chemical MeSH classes in approximately 15 Million publications recorded in PubMed at the time of analysis. The outcome of this analysis revealed that relative to the genome and the MGC collection, this collection is enriched for the presence of genes with published associations with a wide range of diseases and biomedical terms without displaying a particular bias towards any single disease or concept. Thus, this collection is likely to be a powerful resource for researchers who wish to study protein function in a set of genes with documented biomedical significance.

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Related in: MedlinePlus

Workflow diagram of clone production.The entire production process from the design of primers to production of glycerol stocks is shown. The process started by identifying MGC clones in the available plates and then creating array files along with matching PCR primer order files that included two primers anchored at the 3′ end, one for each format. The primers were used to amplify the ORFs from the matching MGC clones. PCR products were monitored in agarose gels, and products were purified prior to capture via In-Fusion reaction. Competent bacterial strains were transformed with the reaction followed by the robotic isolation of 4 resulting colonies per format, which were used to prepare 15% glycerol stocks. Prior to sequencing a single isolate plate of 96 targets were created. As indicated, step specific results were stored in our LIMS.
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pone-0001528-g003: Workflow diagram of clone production.The entire production process from the design of primers to production of glycerol stocks is shown. The process started by identifying MGC clones in the available plates and then creating array files along with matching PCR primer order files that included two primers anchored at the 3′ end, one for each format. The primers were used to amplify the ORFs from the matching MGC clones. PCR products were monitored in agarose gels, and products were purified prior to capture via In-Fusion reaction. Competent bacterial strains were transformed with the reaction followed by the robotic isolation of 4 resulting colonies per format, which were used to prepare 15% glycerol stocks. Prior to sequencing a single isolate plate of 96 targets were created. As indicated, step specific results were stored in our LIMS.

Mentions: The process of converting MGC cDNA clones into ORF clones (Figure 3) was initiated by populating our production tracking database (FLEXGene) with the relevant MGC information, e.g. IMAGE ID, GI number, clone sequence, start/stop of CDS, CDS length, gene information, plate and position in IMAGE/MGC collection. All ORFs were normalized to start with ATG, and natural stop codons either to TAG, or, for the format without C-terminal stop, to TTG (Leu). PCR amplicons were gel purified and captured using the In-Fusion™ enzyme into a modified recombinational cloning vector, pGWNcoXho, which increased the efficiency of capturing DNA fragments larger than 1.5 kb [16]. After transformation into E. coli, constructs were clonally selected and isolated. In total, we successfully produced clonal glycerol stocks for 3,528 of 3,557 targeted genes, an overall success rate of ∼98% (Table 3).


A biomedically enriched collection of 7000 human ORF clones.

Rolfs A, Hu Y, Ebert L, Hoffmann D, Zuo D, Ramachandran N, Raphael J, Kelley F, McCarron S, Jepson DA, Shen B, Baqui MM, Pearlberg J, Taycher E, DeLoughery C, Hoerlein A, Korn B, LaBaer J - PLoS ONE (2008)

Workflow diagram of clone production.The entire production process from the design of primers to production of glycerol stocks is shown. The process started by identifying MGC clones in the available plates and then creating array files along with matching PCR primer order files that included two primers anchored at the 3′ end, one for each format. The primers were used to amplify the ORFs from the matching MGC clones. PCR products were monitored in agarose gels, and products were purified prior to capture via In-Fusion reaction. Competent bacterial strains were transformed with the reaction followed by the robotic isolation of 4 resulting colonies per format, which were used to prepare 15% glycerol stocks. Prior to sequencing a single isolate plate of 96 targets were created. As indicated, step specific results were stored in our LIMS.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211400&req=5

pone-0001528-g003: Workflow diagram of clone production.The entire production process from the design of primers to production of glycerol stocks is shown. The process started by identifying MGC clones in the available plates and then creating array files along with matching PCR primer order files that included two primers anchored at the 3′ end, one for each format. The primers were used to amplify the ORFs from the matching MGC clones. PCR products were monitored in agarose gels, and products were purified prior to capture via In-Fusion reaction. Competent bacterial strains were transformed with the reaction followed by the robotic isolation of 4 resulting colonies per format, which were used to prepare 15% glycerol stocks. Prior to sequencing a single isolate plate of 96 targets were created. As indicated, step specific results were stored in our LIMS.
Mentions: The process of converting MGC cDNA clones into ORF clones (Figure 3) was initiated by populating our production tracking database (FLEXGene) with the relevant MGC information, e.g. IMAGE ID, GI number, clone sequence, start/stop of CDS, CDS length, gene information, plate and position in IMAGE/MGC collection. All ORFs were normalized to start with ATG, and natural stop codons either to TAG, or, for the format without C-terminal stop, to TTG (Leu). PCR amplicons were gel purified and captured using the In-Fusion™ enzyme into a modified recombinational cloning vector, pGWNcoXho, which increased the efficiency of capturing DNA fragments larger than 1.5 kb [16]. After transformation into E. coli, constructs were clonally selected and isolated. In total, we successfully produced clonal glycerol stocks for 3,528 of 3,557 targeted genes, an overall success rate of ∼98% (Table 3).

Bottom Line: Second, clones were selected to represent the best available GenBank reference sequence.The target gene list was compared with 4000 human diseases and over 8500 biological and chemical MeSH classes in approximately 15 Million publications recorded in PubMed at the time of analysis.The outcome of this analysis revealed that relative to the genome and the MGC collection, this collection is enriched for the presence of genes with published associations with a wide range of diseases and biomedical terms without displaying a particular bias towards any single disease or concept.

View Article: PubMed Central - PubMed

Affiliation: Harvard Institute of Proteomics, Harvard Medical School, Cambridge, Massachusetts, USA.

ABSTRACT
We report the production and availability of over 7000 fully sequence verified plasmid ORF clones representing over 3400 unique human genes. These ORF clones were derived using the human MGC collection as template and were produced in two formats: with and without stop codons. Thus, this collection supports the production of either native protein or proteins with fusion tags added to either or both ends. The template clones used to generate this collection were enriched in three ways. First, gene redundancy was removed. Second, clones were selected to represent the best available GenBank reference sequence. Finally, a literature-based software tool was used to evaluate the list of target genes to ensure that it broadly reflected biomedical research interests. The target gene list was compared with 4000 human diseases and over 8500 biological and chemical MeSH classes in approximately 15 Million publications recorded in PubMed at the time of analysis. The outcome of this analysis revealed that relative to the genome and the MGC collection, this collection is enriched for the presence of genes with published associations with a wide range of diseases and biomedical terms without displaying a particular bias towards any single disease or concept. Thus, this collection is likely to be a powerful resource for researchers who wish to study protein function in a set of genes with documented biomedical significance.

Show MeSH
Related in: MedlinePlus