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.
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References
Sessions, A., Burke, E., Presting, G., et al. (2002) A high-throughput Arabidopsis reverse genetics system. Plant Cell 14, 2985–2994.
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.
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.
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.
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.
McElver, J., Tzafrir, I., Aux, G., et al. (2001) Insertional mutagenesis of genes required for seed development in Arabidopsis thaliana. Genetics 159, 1751–1763.
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.
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.
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.
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.
Labarca, C. and Paigen, K. (1980) A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 102, 344–352.
Mazars, G.-R., Moyret, C., Jeanteur, P., and Theillet, C.-G. (1991) Direct sequencing by thermal asymmetric PCR. Nucleic Acids Res. 19, 4783.
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.
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.
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.
Krizkova, L. and Hrouda, M. (1998) Direct repeats of T-DNA integrated in tobacco chromosome: characterization of junction regions. Plant J. 16, 673–680.
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.
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.
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.
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© 2003 Humana Press Inc., Totowa, NJ
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Singer, T., Burke, E. (2003). High-Throughput TAIL-PCR as a Tool to Identify DNA Flanking Insertions. In: Grotewold, E. (eds) Plant Functional Genomics. Methods in Molecular Biology™, vol 236. Humana Press. https://doi.org/10.1385/1-59259-413-1:241
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DOI: https://doi.org/10.1385/1-59259-413-1:241
Publisher Name: Humana Press
Print ISBN: 978-1-58829-145-5
Online ISBN: 978-1-59259-413-9
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