Search tips
Search criteria 


Logo of narLink to Publisher's site
Nucleic Acids Res. 1990 August 25; 18(16): 4833–4842.
PMCID: PMC331957

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


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

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (2.1M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Russel M, Kidd S, Kelley MR. An improved filamentous helper phage for generating single-stranded plasmid DNA. Gene. 1986;45(3):333–338. [PubMed]
  • Chang AC, Cohen SN. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. [PMC free article] [PubMed]
  • Sive HL, St John T. A simple subtractive hybridization technique employing photoactivatable biotin and phenol extraction. Nucleic Acids Res. 1988 Nov 25;16(22):10937–10937. [PMC free article] [PubMed]
  • Dower WJ, Miller JF, Ragsdale CW. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. [PMC free article] [PubMed]
  • Gubler U, Hoffman BJ. A simple and very efficient method for generating cDNA libraries. Gene. 1983 Nov;25(2-3):263–269. [PubMed]
  • Okayama H, Berg P. High-efficiency cloning of full-length cDNA. Mol Cell Biol. 1982 Feb;2(2):161–170. [PMC free article] [PubMed]
  • Han JH, Stratowa C, Rutter WJ. 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. [PubMed]
  • Hoopes BC, McClure WR. Studies on the selectivity of DNA precipitation by spermine. Nucleic Acids Res. 1981 Oct 24;9(20):5493–5504. [PMC free article] [PubMed]
  • Tonnelle C, DeMars R, Long EO. DO beta: a new beta chain gene in HLA-D with a distinct regulation of expression. EMBO J. 1985 Nov;4(11):2839–2847. [PubMed]
  • Meissner PS, Sisk WP, Berman ML. Bacteriophage lambda cloning system for the construction of directional cDNA libraries. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4171–4175. [PubMed]
  • Eun HM, Yoon JW. A simple and versatile method for the preparation of vector-primers by adapter-end-primer ligation. Biotechniques. 1989 Oct;7(9):992–997. [PubMed]
  • Hedrick SM, Cohen DI, Nielsen EA, Davis MM. Isolation of cDNA clones encoding T cell-specific membrane-associated proteins. Nature. 1984 Mar 8;308(5955):149–153. [PubMed]
  • Rhyner TA, Biguet NF, Berrard S, Borbély AA, 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. [PubMed]
  • Miller FD, Naus CC, Higgins GA, Bloom FE, Milner RJ. Developmentally regulated rat brain mRNAs: molecular and anatomical characterization. J Neurosci. 1987 Aug;7(8):2433–2444. [PubMed]
  • Clayton DF, Huecas ME, Sinclair-Thompson EY, Nastiuk KL, Nottebohm F. Probes for rare mRNAs reveal distributed cell subsets in canary brain. Neuron. 1988 May;1(3):249–261. [PubMed]
  • Duguid JR, Rohwer RG, 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. [PubMed]
  • Richter K, Grunz H, Dawid IB. Gene expression in the embryonic nervous system of Xenopus laevis. Proc Natl Acad Sci U S A. 1988 Nov;85(21):8086–8090. [PubMed]
  • Travis GH, Sutcliffe JG. 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. [PubMed]
  • Duguid JR, Bohmont CW, Liu NG, Tourtellotte WW. Changes in brain gene expression shared by scrapie and Alzheimer disease. Proc Natl Acad Sci U S A. 1989 Sep;86(18):7260–7264. [PubMed]
  • Beck E, Ludwig G, Auerswald EA, Reiss B, Schaller H. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene. 1982 Oct;19(3):327–336. [PubMed]
  • Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. [PubMed]
  • 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. [PubMed]
  • Vieira J, Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. [PubMed]

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