Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1990 Aug 25;18(16):4833–4842. doi: 10.1093/nar/18.16.4833

Subtractive hybridization system using single-stranded phagemids with directional inserts.

J L Rubenstein 1, A E Brice 1, R D Ciaranello 1, D Denney 1, M H Porteus 1, T B Usdin 1
PMCID: PMC331957  PMID: 2168539

Abstract

We describe a subtractive hybridization protocol which is designed to permit subtractions between cDNA libraries. The method uses single-stranded phagemids with directional inserts as both the driver and the target. We modified the M13 phagemid vector pBluescript for the directional cDNA cloning and subtractive hybridization. Two simplified methods for efficient construction of directional cDNA libraries are also described. Using a model system, we found that one round of subtractive hybridization results in a 5,000-fold specific subtraction of abundant molecules. We used two methods to quantify the efficiency and verify the specificity of the subtraction. In order to obtain these subtraction efficiencies, it was necessary to develop a method to purify the single-stranded DNA to homogeneity. The single-stranded purification involved using potassium iodide (KI) density centrifugation, restriction endonuclease digestion and phenol extraction in the presence of magnesium. We describe the several advantages of using directional inserts for the subtraction procedure.

Full text

PDF
4833

Images in this article

4837Image
on p.4837

Selected References

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

  1. Beck E., Ludwig G., Auerswald E. A., Reiss B., Schaller H. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene. 1982 Oct;19(3):327–336. doi: 10.1016/0378-1119(82)90023-3. [DOI] [PubMed] [Google Scholar]
  2. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Clayton D. F., Huecas M. E., Sinclair-Thompson E. Y., Nastiuk K. L., Nottebohm F. Probes for rare mRNAs reveal distributed cell subsets in canary brain. Neuron. 1988 May;1(3):249–261. doi: 10.1016/0896-6273(88)90146-8. [DOI] [PubMed] [Google Scholar]
  4. Dower W. J., Miller J. F., Ragsdale C. W. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. doi: 10.1093/nar/16.13.6127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Duguid J. R., Bohmont C. W., Liu N. G., Tourtellotte W. W. Changes in brain gene expression shared by scrapie and Alzheimer disease. Proc Natl Acad Sci U S A. 1989 Sep;86(18):7260–7264. doi: 10.1073/pnas.86.18.7260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Duguid J. R., Rohwer R. G., Seed B. Isolation of cDNAs of scrapie-modulated RNAs by subtractive hybridization of a cDNA library. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5738–5742. doi: 10.1073/pnas.85.15.5738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eun H. M., Yoon J. W. A simple and versatile method for the preparation of vector-primers by adapter-end-primer ligation. Biotechniques. 1989 Oct;7(9):992-4, 996-7. [PubMed] [Google Scholar]
  8. Gubler U., Hoffman B. J. A simple and very efficient method for generating cDNA libraries. Gene. 1983 Nov;25(2-3):263–269. doi: 10.1016/0378-1119(83)90230-5. [DOI] [PubMed] [Google Scholar]
  9. Han J. H., Stratowa C., Rutter W. J. Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning. Biochemistry. 1987 Mar 24;26(6):1617–1625. doi: 10.1021/bi00380a020. [DOI] [PubMed] [Google Scholar]
  10. Hedrick S. M., Cohen D. I., Nielsen E. A., Davis M. M. Isolation of cDNA clones encoding T cell-specific membrane-associated proteins. Nature. 1984 Mar 8;308(5955):149–153. doi: 10.1038/308149a0. [DOI] [PubMed] [Google Scholar]
  11. Hoopes B. C., McClure W. R. Studies on the selectivity of DNA precipitation by spermine. Nucleic Acids Res. 1981 Oct 24;9(20):5493–5504. doi: 10.1093/nar/9.20.5493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Meissner P. S., Sisk W. P., Berman M. L. Bacteriophage lambda cloning system for the construction of directional cDNA libraries. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4171–4175. doi: 10.1073/pnas.84.12.4171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  14. Miller F. D., Naus C. C., Higgins G. A., Bloom F. E., Milner R. J. Developmentally regulated rat brain mRNAs: molecular and anatomical characterization. J Neurosci. 1987 Aug;7(8):2433–2444. [PMC free article] [PubMed] [Google Scholar]
  15. Okayama H., Berg P. High-efficiency cloning of full-length cDNA. Mol Cell Biol. 1982 Feb;2(2):161–170. doi: 10.1128/mcb.2.2.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rhyner T. A., Biguet N. F., Berrard S., Borbély A. A., Mallet J. An efficient approach for the selective isolation of specific transcripts from complex brain mRNA populations. J Neurosci Res. 1986;16(1):167–181. doi: 10.1002/jnr.490160116. [DOI] [PubMed] [Google Scholar]
  17. Richter K., Grunz H., Dawid I. B. Gene expression in the embryonic nervous system of Xenopus laevis. Proc Natl Acad Sci U S A. 1988 Nov;85(21):8086–8090. doi: 10.1073/pnas.85.21.8086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Russel M., Kidd S., Kelley M. R. An improved filamentous helper phage for generating single-stranded plasmid DNA. Gene. 1986;45(3):333–338. doi: 10.1016/0378-1119(86)90032-6. [DOI] [PubMed] [Google Scholar]
  19. Sive H. L., St John T. A simple subtractive hybridization technique employing photoactivatable biotin and phenol extraction. Nucleic Acids Res. 1988 Nov 25;16(22):10937–10937. doi: 10.1093/nar/16.22.10937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tonnelle C., DeMars R., Long E. O. DO beta: a new beta chain gene in HLA-D with a distinct regulation of expression. EMBO J. 1985 Nov;4(11):2839–2847. doi: 10.1002/j.1460-2075.1985.tb04012.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Travis G. H., Sutcliffe J. G. Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of low-abundance monkey cortex-specific mRNAs. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1696–1700. doi: 10.1073/pnas.85.5.1696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  23. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES