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. 1997 May 1;25(9):1830–1835. doi: 10.1093/nar/25.9.1830

Primer design for a prokaryotic differential display RT-PCR.

R Fislage 1, M Berceanu 1, Y Humboldt 1, M Wendt 1, H Oberender 1
PMCID: PMC146661  PMID: 9108168

Abstract

We have developed a primer set for a prokaryotic differential display of mRNA in the Enterobacteriaceae group. Each combination of ten 10mer and ten 11mer primers generates up to 85 bands from total Escherichia coli RNA, thus covering expressed sequences of a complete bacterial genome. Due to the lack of polyadenylation in prokaryotic RNA the type T11VN anchored oligonucleotides for the reverse transcriptase reaction had to be replaced with respect to the original method described by Liang and Pardee [ Science , 257, 967-971 (1992)]. Therefore, the sequences of both the 10mer and the new 11mer oligonucleotides were determined by a statistical evaluation of species-specific coding regions extracted from the EMBL database. The 11mer primers used for reverse transcription were selected for localization in the 3'-region of the bacterial RNA. The 10mer primers preferentially bind to the 5'-end of the RNA. None of the primers show homology to rRNA or other abundant small RNA species. Randomly sampled cDNA bands were checked for their bacterial origin either by re-amplification, cloning and sequencing or by re-amplification and direct sequencing with 10mer and 11mer primers after asymmetric PCR.

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Selected References

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  1. Abu Kwaik Y., Pederson L. L. The use of differential display-PCR to isolate and characterize a Legionella pneumophila locus induced during the intracellular infection of macrophages. Mol Microbiol. 1996 Aug;21(3):543–556. doi: 10.1111/j.1365-2958.1996.tb02563.x. [DOI] [PubMed] [Google Scholar]
  2. Bauer D., Müller H., Reich J., Riedel H., Ahrenkiel V., Warthoe P., Strauss M. Identification of differentially expressed mRNA species by an improved display technique (DDRT-PCR). Nucleic Acids Res. 1993 Sep 11;21(18):4272–4280. doi: 10.1093/nar/21.18.4272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bult C. J., White O., Olsen G. J., Zhou L., Fleischmann R. D., Sutton G. G., Blake J. A., FitzGerald L. M., Clayton R. A., Gocayne J. D. Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science. 1996 Aug 23;273(5278):1058–1073. doi: 10.1126/science.273.5278.1058. [DOI] [PubMed] [Google Scholar]
  4. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  5. Crampton J. M., Davies K. E., Knapp T. F. The occurrence of families of repetitive sequences in a library of cloned cDNA from human lymphocytes. Nucleic Acids Res. 1981 Aug 11;9(15):3821–3834. doi: 10.1093/nar/9.15.3821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Crowe J. S., Cooper H. J., Smith M. A., Sims M. J., Parker D., Gewert D. Improved cloning efficiency of polymerase chain reaction (PCR) products after proteinase K digestion. Nucleic Acids Res. 1991 Jan 11;19(1):184–184. doi: 10.1093/nar/19.1.184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Etzold T., Argos P. SRS--an indexing and retrieval tool for flat file data libraries. Comput Appl Biosci. 1993 Feb;9(1):49–57. doi: 10.1093/bioinformatics/9.1.49. [DOI] [PubMed] [Google Scholar]
  8. Fleischmann R. D., Adams M. D., White O., Clayton R. A., Kirkness E. F., Kerlavage A. R., Bult C. J., Tomb J. F., Dougherty B. A., Merrick J. M. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995 Jul 28;269(5223):496–512. doi: 10.1126/science.7542800. [DOI] [PubMed] [Google Scholar]
  9. Fraser C. M., Gocayne J. D., White O., Adams M. D., Clayton R. A., Fleischmann R. D., Bult C. J., Kerlavage A. R., Sutton G., Kelley J. M. The minimal gene complement of Mycoplasma genitalium. Science. 1995 Oct 20;270(5235):397–403. doi: 10.1126/science.270.5235.397. [DOI] [PubMed] [Google Scholar]
  10. Gyllensten U. B., Erlich H. A. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7652–7656. doi: 10.1073/pnas.85.20.7652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kaneko T., Sato S., Kotani H., Tanaka A., Asamizu E., Nakamura Y., Miyajima N., Hirosawa M., Sugiura M., Sasamoto S. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res. 1996 Jun 30;3(3):109–136. doi: 10.1093/dnares/3.3.109. [DOI] [PubMed] [Google Scholar]
  12. Lawlis V. B., Dennis M. S., Chen E. Y., Smith D. H., Henner D. J. Cloning and sequencing of the xylose isomerase and xylulose kinase genes of Escherichia coli. Appl Environ Microbiol. 1984 Jan;47(1):15–21. doi: 10.1128/aem.47.1.15-21.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Liang P., Pardee A. B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science. 1992 Aug 14;257(5072):967–971. doi: 10.1126/science.1354393. [DOI] [PubMed] [Google Scholar]
  14. Schellenberg G. D., Sarthy A., Larson A. E., Backer M. P., Crabb J. W., Lidstrom M., Hall B. D., Furlong C. E. Xylose isomerase from Escherichia coli. Characterization of the protein and the structural gene. J Biol Chem. 1984 Jun 10;259(11):6826–6832. [PubMed] [Google Scholar]
  15. Schlösser A., Hamann A., Bossemeyer D., Schneider E., Bakker E. P. NAD+ binding to the Escherichia coli K(+)-uptake protein TrkA and sequence similarity between TrkA and domains of a family of dehydrogenases suggest a role for NAD+ in bacterial transport. Mol Microbiol. 1993 Aug;9(3):533–543. doi: 10.1111/j.1365-2958.1993.tb01714.x. [DOI] [PubMed] [Google Scholar]
  16. Welsh J., McClelland M. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 1990 Dec 25;18(24):7213–7218. doi: 10.1093/nar/18.24.7213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Williams J. G., Kubelik A. R., Livak K. J., Rafalski J. A., Tingey S. V. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 1990 Nov 25;18(22):6531–6535. doi: 10.1093/nar/18.22.6531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Wong K. K., McClelland M. Stress-inducible gene of Salmonella typhimurium identified by arbitrarily primed PCR of RNA. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):639–643. doi: 10.1073/pnas.91.2.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Xu J., Johnson R. C. aldB, an RpoS-dependent gene in Escherichia coli encoding an aldehyde dehydrogenase that is repressed by Fis and activated by Crp. J Bacteriol. 1995 Jun;177(11):3166–3175. doi: 10.1128/jb.177.11.3166-3175.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Yoshikawa A., Isono S., Sheback A., Isono K. Cloning and nucleotide sequencing of the genes rimI and rimJ which encode enzymes acetylating ribosomal proteins S18 and S5 of Escherichia coli K12. Mol Gen Genet. 1987 Oct;209(3):481–488. doi: 10.1007/BF00331153. [DOI] [PubMed] [Google Scholar]

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