TY - JOUR
T1 - ORF-FINDER
T2 - A vector for high-throughput gene identification
AU - Rombel, Irene T.
AU - Sykes, Kathryn F.
AU - Rayner, Simon
AU - Johnston, Stephen Albert
N1 - Funding Information:
We would like to thank Drs. Tobias Schlupp and Rick Tarleton for the kind gifts of N. caninum and T. cruzi genomic DNA, respectively. This work was supported by a grant from DARPA to S.A.J. and an Advanced Technology Program grant from the Texas Higher Education Coordinating Board to I.T.R.
PY - 2002/1/9
Y1 - 2002/1/9
N2 - We have developed a simple and efficient system (ORF-FINDER) for selecting open reading frames (ORFs) from randomly fragmented genomic DNA fragments. The ORF-FINDER vectors are plasmids that contain a translational start site out of frame with respect to the gene for green fluorescent protein (GFP). Insertion of DNA fragments that bring the initiating ATG in frame with GFP and that contain no stop codons (that is, ORFs) results in the expression of ORF-GFP fusion proteins. In addition, we have developed software (GeneWorks and GenomeAnalyzer) to predict the optimal insert size for maximizing the number of gene-coding ORFs and minimizing unintentionally selected non-coding ORFs. To demonstrate the feasibility of using the ORF-FINDER system to screen genomes for ORFs, we cloned yeast genomic DNA and succeeded in enriching for ORFs by 25-fold. Furthermore, we have shown that the vector can effectively isolate ORFs from the more complex genomes of eukaryotic parasites. We envision that ORF-FINDER will have several applications including genome sequencing projects, gene building from oligonucleotides and construction of expression libraries enriched for ORFs.
AB - We have developed a simple and efficient system (ORF-FINDER) for selecting open reading frames (ORFs) from randomly fragmented genomic DNA fragments. The ORF-FINDER vectors are plasmids that contain a translational start site out of frame with respect to the gene for green fluorescent protein (GFP). Insertion of DNA fragments that bring the initiating ATG in frame with GFP and that contain no stop codons (that is, ORFs) results in the expression of ORF-GFP fusion proteins. In addition, we have developed software (GeneWorks and GenomeAnalyzer) to predict the optimal insert size for maximizing the number of gene-coding ORFs and minimizing unintentionally selected non-coding ORFs. To demonstrate the feasibility of using the ORF-FINDER system to screen genomes for ORFs, we cloned yeast genomic DNA and succeeded in enriching for ORFs by 25-fold. Furthermore, we have shown that the vector can effectively isolate ORFs from the more complex genomes of eukaryotic parasites. We envision that ORF-FINDER will have several applications including genome sequencing projects, gene building from oligonucleotides and construction of expression libraries enriched for ORFs.
KW - Expression library immunization
KW - Functional genomic screen
KW - Open reading frame
KW - Open reading frame selection vector
KW - Parasite
UR - http://www.scopus.com/inward/record.url?scp=0037045398&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0037045398&partnerID=8YFLogxK
U2 - 10.1016/S0378-1119(01)00819-8
DO - 10.1016/S0378-1119(01)00819-8
M3 - Article
C2 - 11814675
AN - SCOPUS:0037045398
VL - 282
SP - 33
EP - 41
JO - Gene
JF - Gene
SN - 0378-1119
IS - 1-2
ER -