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Walk-PCR based isolation of regions flanking

the left border of T-DNA inserts

Isolation of PCR fragments is performed using an adapter-ligation PCR method (1) also called PCR –walking (2), and primers specific to the sequence at the T-DNA left border. This sequence remains identical across the 3 different T-DNA constructs used to generate the library (Figure 1). This technique relies on the use of blunt-end restriction enzymes followed by a ligation of asymmetric adapters (see Figure 2 and Table 1). For each primary transformant, genomic DNA is independently digested by DraI and SspI restriction enzymes. The PCR fragments are isolated following two PCR runs (i.e. PCR1 and PCR2 « Nested PCRs »). The PCR2 amplification products exhibiting a sharp bands following agarose gel electrophoresis are directly sequenced using  a third primer CAMB6. The sequencing has been accomplished by M. Delseny laboratory (CNRS, U. Perpignan) for the first 1,000 sequences, then by GénomeExpress or Qiagen companies (Sallaud et al (2004), in press).  The resulting sequences are attributed a sequencing code DXXYXY and SXXYXY for PCR fragments amplified from DraI and SspI digests respectively. 

Table 1 : Primers used for isolating and sequencing the regions flanking the left border

of T-DNA inserts in the Génoplante rice insertion line library.

Walk-PCR based isolation of regions flanking

the right border of T-DNA inserts

to appear soon

Acknowledgements 

The team wishes to warmly thank Monique Raynal, Martine Devic and Michel Delseny (CNRS, Université de Perpignan) as well as Sandrine Balzergue, Bertrand Dubreucq  and Loic Lepiniec (URGV, INRA, Evry) for their useful advices at the initiation of this research project of FST isolation and sequencing. As to bioninformatics, we used software created by Véronique Bruneau, Franck Sanson, and Alain Lecharny (URGV, INRA, Evry). The p4955, p4956, p4978, p4984, p4985 and p4987 have been prepared by the team of Andreas Betzner (Biogemma, ANU, Austratia).  

References

¹ Siebert et al., NAR (1995) 23:1087-1088

² Devic et al., Plant Physiol Biochem, (1997) 35 :331-339

³ Balzergue et al., Biotechnique (2001) 30 :496-504