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. 1995 Mar 14;92(6):2209–2213. doi: 10.1073/pnas.92.6.2209

Systematic screening of an arrayed cDNA library by PCR.

D J Munroe 1, R Loebbert 1, E Bric 1, T Whitton 1, D Prawitt 1, D Vu 1, A Buckler 1, A Winterpacht 1, B Zabel 1, D E Housman 1
PMCID: PMC42453  PMID: 7892249

Abstract

We have developed a PCR-based method for rapid and effective screening of arrayed cDNA libraries. This strategy directly addresses the limitations of conventional hybridization-based schemes and provides a more rapid, cost-effective, and sensitive method compatible with large-scale and routine cDNA clone recovery. To prepare arrayed libraries, 1-2 x 10(6) cDNA clones were propagated as individual plaques on solid medium in 24-well culture dishes at approximately 250 plaque-forming units per well. Phage suspensions were prepared from each well and transferred to a 96-well format. To screen the library, pools were generated that correspond to each individual 96-well plate and to each row and column within "blocks" of six plates each. Library screening for specific cDNA clones was conducted in a systematic and hierarchical fashion beginning with the plate pools. Next, the row/column pools corresponding to each positive plate pool were screened. Finally, isolated clones from within each positive well were identified by hybridization. We have applied this approach to the screening of an arrayed human brain cDNA library resulting in the recovery of cDNAs corresponding to > 25 genes and expressed sequence tags.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams M. D., Kelley J. M., Gocayne J. D., Dubnick M., Polymeropoulos M. H., Xiao H., Merril C. R., Wu A., Olde B., Moreno R. F. Complementary DNA sequencing: expressed sequence tags and human genome project. Science. 1991 Jun 21;252(5013):1651–1656. doi: 10.1126/science.2047873. [DOI] [PubMed] [Google Scholar]
  2. Adams M. D., Kerlavage A. R., Fields C., Venter J. C. 3,400 new expressed sequence tags identify diversity of transcripts in human brain. Nat Genet. 1993 Jul;4(3):256–267. doi: 10.1038/ng0793-256. [DOI] [PubMed] [Google Scholar]
  3. Ambrose C., James M., Barnes G., Lin C., Bates G., Altherr M., Duyao M., Groot N., Church D., Wasmuth J. J. A novel G protein-coupled receptor kinase gene cloned from 4p16.3. Hum Mol Genet. 1992 Dec;1(9):697–703. doi: 10.1093/hmg/1.9.697. [DOI] [PubMed] [Google Scholar]
  4. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  5. Bronner C. E., Baker S. M., Morrison P. T., Warren G., Smith L. G., Lescoe M. K., Kane M., Earabino C., Lipford J., Lindblom A. Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer. Nature. 1994 Mar 17;368(6468):258–261. doi: 10.1038/368258a0. [DOI] [PubMed] [Google Scholar]
  6. Broome S., Gilbert W. Immunological screening method to detect specific translation products. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2746–2749. doi: 10.1073/pnas.75.6.2746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Buckler A. J., Chang D. D., Graw S. L., Brook J. D., Haber D. A., Sharp P. A., Housman D. E. Exon amplification: a strategy to isolate mammalian genes based on RNA splicing. Proc Natl Acad Sci U S A. 1991 May 1;88(9):4005–4009. doi: 10.1073/pnas.88.9.4005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Church D. M., Banks L. T., Rogers A. C., Graw S. L., Housman D. E., Gusella J. F., Buckler A. J. Identification of human chromosome 9 specific genes using exon amplification. Hum Mol Genet. 1993 Nov;2(11):1915–1920. doi: 10.1093/hmg/2.11.1915. [DOI] [PubMed] [Google Scholar]
  9. Church D. M., Stotler C. J., Rutter J. L., Murrell J. R., Trofatter J. A., Buckler A. J. Isolation of genes from complex sources of mammalian genomic DNA using exon amplification. Nat Genet. 1994 Jan;6(1):98–105. doi: 10.1038/ng0194-98. [DOI] [PubMed] [Google Scholar]
  10. Duyao M. P., Taylor S. A., Buckler A. J., Ambrose C. M., Lin C., Groot N., Church D., Barnes G., Wasmuth J. J., Housman D. E. A gene from chromosome 4p16.3 with similarity to a superfamily of transporter proteins. Hum Mol Genet. 1993 Jun;2(6):673–676. doi: 10.1093/hmg/2.6.673. [DOI] [PubMed] [Google Scholar]
  11. Duyk G. M., Kim S. W., Myers R. M., Cox D. R. Exon trapping: a genetic screen to identify candidate transcribed sequences in cloned mammalian genomic DNA. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8995–8999. doi: 10.1073/pnas.87.22.8995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Erlich H. A., Cohen S. N., McDevitt H. O. A sensitive radioimmunoassay for detecting products translated from cloned DNA fragments. Cell. 1978 Apr;13(4):681–689. doi: 10.1016/0092-8674(78)90218-0. [DOI] [PubMed] [Google Scholar]
  13. Glaser T., Housman D., Lewis W. H., Gerhard D., Jones C. A fine-structure deletion map of human chromosome 11p: analysis of J1 series hybrids. Somat Cell Mol Genet. 1989 Nov;15(6):477–501. doi: 10.1007/BF01534910. [DOI] [PubMed] [Google Scholar]
  14. Green E. D., Olson M. V. Systematic screening of yeast artificial-chromosome libraries by use of the polymerase chain reaction. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1213–1217. doi: 10.1073/pnas.87.3.1213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Higgins M. J., Smilinich N. J., Sait S., Koenig A., Pongratz J., Gessler M., Richard C. W., 3rd, James M. R., Sanford J. P., Kim B. W. An ordered NotI fragment map of human chromosome band 11p15. Genomics. 1994 Sep 1;23(1):211–222. doi: 10.1006/geno.1994.1479. [DOI] [PubMed] [Google Scholar]
  17. Khan A. S., Wilcox A. S., Polymeropoulos M. H., Hopkins J. A., Stevens T. J., Robinson M., Orpana A. K., Sikela J. M. Single pass sequencing and physical and genetic mapping of human brain cDNAs. Nat Genet. 1992 Nov;2(3):180–185. doi: 10.1038/ng1192-180. [DOI] [PubMed] [Google Scholar]
  18. Lovett M., Kere J., Hinton L. M. Direct selection: a method for the isolation of cDNAs encoded by large genomic regions. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9628–9632. doi: 10.1073/pnas.88.21.9628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Parimoo S., Patanjali S. R., Shukla H., Chaplin D. D., Weissman S. M. cDNA selection: efficient PCR approach for the selection of cDNAs encoded in large chromosomal DNA fragments. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9623–9627. doi: 10.1073/pnas.88.21.9623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Patanjali S. R., Parimoo S., Weissman S. M. Construction of a uniform-abundance (normalized) cDNA library. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1943–1947. doi: 10.1073/pnas.88.5.1943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rossi J. M., Burke D. T., Leung J. C., Koos D. S., Chen H., Tilghman S. M. Genomic analysis using a yeast artificial chromosome library with mouse DNA inserts. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2456–2460. doi: 10.1073/pnas.89.6.2456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sasaki Y. F., Ayusawa D., Oishi M. Construction of a normalized cDNA library by introduction of a semi-solid mRNA-cDNA hybridization system. Nucleic Acids Res. 1994 Mar 25;22(6):987–992. doi: 10.1093/nar/22.6.987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Shepherd N. S., Pfrogner B. D., Coulby J. N., Ackerman S. L., Vaidyanathan G., Sauer R. H., Balkenhol T. C., Sternberg N. Preparation and screening of an arrayed human genomic library generated with the P1 cloning system. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2629–2633. doi: 10.1073/pnas.91.7.2629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Trofatter J. A., MacCollin M. M., Rutter J. L., Murrell J. R., Duyao M. P., Parry D. M., Eldridge R., Kley N., Menon A. G., Pulaski K. A novel moesin-, ezrin-, radixin-like gene is a candidate for the neurofibromatosis 2 tumor suppressor. Cell. 1993 Mar 12;72(5):791–800. doi: 10.1016/0092-8674(93)90406-g. [DOI] [PubMed] [Google Scholar]
  26. Vidal S. M., Malo D., Vogan K., Skamene E., Gros P. Natural resistance to infection with intracellular parasites: isolation of a candidate for Bcg. Cell. 1993 May 7;73(3):469–485. doi: 10.1016/0092-8674(93)90135-d. [DOI] [PubMed] [Google Scholar]
  27. Vulpe C., Levinson B., Whitney S., Packman S., Gitschier J. Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transporting ATPase. Nat Genet. 1993 Jan;3(1):7–13. doi: 10.1038/ng0193-7. [DOI] [PubMed] [Google Scholar]
  28. Wilcox A. S., Khan A. S., Hopkins J. A., Sikela J. M. Use of 3' untranslated sequences of human cDNAs for rapid chromosome assignment and conversion to STSs: implications for an expression map of the genome. Nucleic Acids Res. 1991 Apr 25;19(8):1837–1843. doi: 10.1093/nar/19.8.1837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Young R. A., Davis R. W. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. doi: 10.1073/pnas.80.5.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]

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