Skip to main content

PCR-based detection of a rare linear DNA in cell culture


The described method allows for detection of rare linear DNA fragments generated during genomic deletions. The predicted limit of the detection is one DNA molecule per 107 or more cells. The method is based on anchor PCR and involves gel separation of the linear DNA fragment and chromosomal DNA before amplification. The detailed chemical structure of the ends of the linear DNA can be defined with the use of additional PCR-based protocols. The method was applied to study the short-lived linear DNA generated during programmed genomic deletions in a ciliate. It can be useful in studies of spontaneous DNA deletions in cell culture or for tracking intracellular modifications at the ends of transfected DNA during gene therapy trials.


  1. 1.

    Paques F, Haber JE. Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiology & Molecular Biology Reviews. 1999; 63(2):349–404.

    CAS  Google Scholar 

  2. 2.

    Yao M-C. Site-specific chromosome breakage and DNA deletions in ciliates In Mobile DNA (ed. D.E. Berg and M.M. Howe), pp. 715–734. American Society for Microbiology, Washington, D.C., 1989.

    Google Scholar 

  3. 3.

    Saveliev SV, Cox MM. Product analysis illuminates the final steps of IES deletion in Tetrahymena thermophila. EMBO J. 2001; 20(12):3251–3261.

    PubMed  Article  CAS  Google Scholar 

  4. 4.

    Saveliev SV, Cox MM. The fate of deleted DNA produced during programmed genomic deletion events in Tetrahymena thermophila. Nucleic Acids Res. 1994; 22:5695–5701.

    PubMed  Article  CAS  Google Scholar 

  5. 5.

    Coyne RS, Nikiforov MA, Smothers JF, Allis CD, Yao MC. Parental Expression of the chromodomain protein Pdd1 is required for completion of programmed DNA elimination and nuclear differentiation. Molecular Cell. 1999; 4:865–872.

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    Austerberry CF, Allis CD, Yao M-C. Specific DNA rearrangements in synchronously developing nuclei of Tetrahymena. Proc. Natl. Acad. Sci. USA 1984; 81:7383–7387.

    PubMed  Article  CAS  Google Scholar 

  7. 7.

    Bruns PJ, Brussard TB. Pair formation in Tetrahymena pyriformis, an inducible developmental system. Journal of Experimental Zoology. 1974; 188(3):337–344.

    PubMed  Article  CAS  Google Scholar 

  8. 8.

    Austerberry CF, Yao M-C. Nucleotide sequence structure and consistency of a developmentally regulated DNA deletion in Tetrahymena thermophila. Mol. Cell Biol. 1987; 7:435–443.

    PubMed  CAS  Google Scholar 

  9. 9.

    Saveliev SV, Cox MM. Developmentally programmed DNA deletion in Tetrahymena thermophila by a transposition-like reaction pathway. Embo J. 1996; 15(11):2858–2869.

    PubMed  CAS  Google Scholar 

  10. 10.

    Mueller PR, Wold B. In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science. 1989; 246:780–786.

    PubMed  Article  CAS  Google Scholar 

  11. 11.

    Sambrook J, Russell DW. Molecular Cloning: A laboratory manual. 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 2001.

    Google Scholar 

  12. 12.

    Clark JM. Novel non-template nucleotide addition reactions catalyzed by prokaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 1988; 16:9677–9686.

    PubMed  Article  CAS  Google Scholar 

  13. 13.

    Dumas JB, Edwards M, Delort J, Mallet J. Oligonucleotide ligation to single-stranded cDNAs: a new tool for cloning 5′ ends of mRNAs and for constructing cDNA libraries by in vitro amplification. Nucleic Acids Res. 1991; 19 (19):5227–5232.

    Article  Google Scholar 

  14. 14.

    Loh EY, Elliott JF, Cwirla S, Lanier LL, Davis MM. Polymerase chain reaction with single-sided specificity: analysis of T cell receptor delta chain. Science. 1989; 243:217–220.

    PubMed  Article  CAS  Google Scholar 

  15. 15.

    Bach S, Buchrieser C, Prentice M, Guiyoule A, Msadek T, Carniel E. The high-pathogenicity island of Yersinia enterocolitica Ye8081 undergoes low-frequency deletion but not precise excision, suggesting recent stabilization in the genome. [Journal Article] Infection & Immunity. 1999; 67(10):5091–5099.

    CAS  Google Scholar 

  16. 16.

    Seyfert HM, Cleffmann G. Mean macronuclear DNA contents are variable in the ciliate Tetrahymena. Journal of Cell Science. 1982; 58:211–223.

    PubMed  CAS  Google Scholar 

  17. 17.

    Mikel UV. Absolute DNA values from Feulgen microspectrophotometric measurements and quantitative electron microscopy: A comparison of two species. Analytical & Quantitative Cytology & Histology. 1987; 9(1):13–16.

    CAS  Google Scholar 

Download references

Author information



Corresponding author

Correspondence to Sergei V. Saveliev.

Additional information

Published: November 11, 2002

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Saveliev, S.V. PCR-based detection of a rare linear DNA in cell culture. Biol Proced Online 4, 70–80 (2002).

Download citation

Indexing terms

  • Polymerase Chain Reaction
  • DNA
  • cell culture