Advertisement

High-Throughput TAIL-PCR as a Tool to Identify DNA Flanking Insertions

  • Tatjana Singer
  • Ellen Burke
Part of the Methods in Molecular Biology™ book series (MIMB, volume 236)

Abstract

Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) is a fast and efficient method to amplify unknown sequences adjacent to known insertion sites in Arabidopsis. Nested, insertion-specific primers are used together with arbitrary degenerate primers (AD primers), which are designed to differ in their annealing temperatures. Alternating cycles of high and low annealing temperature yield specific products bordered by an insertion-specific primer on one side and an AD primer on the other. Further specifity is obtained through subsequent rounds of TAIL-PCR, using nested insertion-specific primers. The increasing availability of whole genome sequences renders TAIL-PCR an attractive tool to easily identify insertion sites in large genome tagging populations through the direct sequencing of TAIL-PCR products. For large-scale functional genomics approaches, it is desirable to obtain flanking sequences for each individual in the population in a fast and cost-effective manner. In this chapter, we describe a TAIL-PCR method amenable for high-throughput production (HT-TAIL-PCR) in Arabidopsis (1). Based on this protocol, HT-TAIL-PCR may be easily adapted for other organisms.

Key Words

TAIL-PCR HT-TAIL-PCR T-DNA transposon Arabidopsis high-throughput reverse genetics tagging population knock-out 

References

  1. 1.
    Sessions, A., Burke, E., Presting, G., et al. (2002) A high-throughput Arabidopsis reverse genetics system. Plant Cell 14, 2985–2994.PubMedCrossRefGoogle Scholar
  2. 2.
    Parinov, S. and Sundaresan, V. (2000) Functional genomics in Arabidopsis: large-scale insertional mutagenesis complements the genome sequencing project. Curr. Opin. Biotechnol. 11, 157–161.PubMedCrossRefGoogle Scholar
  3. 3.
    Altschul, S. F., Madden, T. L., Schaffer, A. A., et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402.PubMedCrossRefGoogle Scholar
  4. 4.
    Liu, Y. G. and Whittier, R. F. (1995) Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25, 674–681.PubMedCrossRefGoogle Scholar
  5. 5.
    Liu, Y. G., Mitsukawa, N., Oosumi, T., and Whittier, R. F. (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J. 8, 457–463.PubMedCrossRefGoogle Scholar
  6. 6.
    McElver, J., Tzafrir, I., Aux, G., et al. (2001) Insertional mutagenesis of genes required for seed development in Arabidopsis thaliana. Genetics 159, 1751–1763.PubMedGoogle Scholar
  7. 7.
    Budziszewski, G. J., Lewis, S. P., Glover, L. W., et al. (2001) Arabidopsis genes essential for seedling viability: isolation of insertional mutants and molecular cloning. Genetics 159, 1765–1778.PubMedGoogle Scholar
  8. 8.
    Parinov, S., Sevugan, M., Ye, D., Yang, W.-C., Kumaran, M., and Sundaresan, V. (1999) Analysis of flanking sequences from dissociation insertion lines. A database for reverse genetics in Arabidopsis. Plant Cell 11, 2263–2270.PubMedCrossRefGoogle Scholar
  9. 9.
    Tsugeki, R., Kochieva, E. Z., and Fedoroff, N. V. (1996) A transposon insertion in the Arabidopsis SSR16 gene causes an embryo-defective lethal mutation. Plant J. 10, 479–489.PubMedCrossRefGoogle Scholar
  10. 10.
    Tissier, A. F., Marillonnet, S., Klimyuk, V., et al. (1999) Multiple independent defective Suppressor-mutator transposon insertions in Arabidopsis: a tool for functional genomics. Plant Cell 11, 1841–1852.PubMedCrossRefGoogle Scholar
  11. 11.
    Labarca, C. and Paigen, K. (1980) A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 102, 344–352.PubMedCrossRefGoogle Scholar
  12. 12.
    Mazars, G.-R., Moyret, C., Jeanteur, P., and Theillet, C.-G. (1991) Direct sequencing by thermal asymmetric PCR. Nucleic Acids Res. 19, 4783.PubMedCrossRefGoogle Scholar
  13. 13.
    Gheysen, G., Herman, L., Breyne, P., Gielen, J., Van Montagu, M., and Depicker, A. (1990) Cloning and sequence analysis of truncated T-DNA inserts from Nicotiana tabacum. Gene 94, 155–163.PubMedCrossRefGoogle Scholar
  14. 14.
    Nacry, P., Camilleri, C., Courtial, B., Caboche, M., and Bouchez, D. (1998) Major chromosomal rearrangements induced by T-DNA transformation in Arabidopsis. Genetics 149, 641–650.PubMedGoogle Scholar
  15. 15.
    De Neve, M., De Buck, S., Jacobs, A., Van Montagu, M., and Depicker, A. (1997) T-DNA integration patterns in co-transformed plant cells suggest that T-DNA repeats originate from cointegration of separate T-DNAs. Plant J. 11, 15–29.PubMedCrossRefGoogle Scholar
  16. 16.
    Krizkova, L. and Hrouda, M. (1998) Direct repeats of T-DNA integrated in tobacco chromosome: characterization of junction regions. Plant J. 16, 673–680.PubMedCrossRefGoogle Scholar
  17. 17.
    De Buck, S., Jacobs, A., Van Montagu, M., and Depicker, A. (1999) The DNA sequences of T-DNA junctions suggest that complex T-DNA loci are formed by a recombination process resembling T-DNA integration. Plant J. 20, 295–304.PubMedCrossRefGoogle Scholar
  18. 18.
    Ewing, B., Hillier, L., Wendl, M., and Green, P. (1998) Basecalling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 8, 175–185.PubMedGoogle Scholar
  19. 19.
    Grossniklaus, U., Vielle-Calzada, J. P., Hoeppner, M. A., and Gagliano, W. B. (1998) Maternal control of embryogenesis by MEDEA, a polycomb group gene in Arabidopsis. Science 280, 446–450.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2003

Authors and Affiliations

  • Tatjana Singer
    • 1
  • Ellen Burke
    • 1
  1. 1.Diversa CorporationSan Diego

Personalised recommendations