MFS Study
Maize Mu transposon flanking sequences
DLA, a genome walking method, was adapted for sequencing Mu flanking sequences using the 454 FLX platform.  Mu flanking sequences from different Mu lines were amplified by a genome walking method, DLA (Liu et al., Genetics, Submitted) and pooled for the 454 pyrosequencing. The primers used to construct the DNA library are composite primers. DNA barcodes (6 bp) were inserted between the 454 primer A and the Mu-specific primer to allow different input samples that were pooled in the same 454 run to be distinguished after sequencing. The resulting library amplified with Primer 1 and Primer 2 was subjected to 454 sequencing using 454 primer A. Totally, Gl2 and Mu2 samples were subjected to sequencing. Each experiences two technical replication. Each replication was run in the half plate.
The Mu transposon system of maize is highly active, with each of the ~50-100 copies transposing on average once each generation.  The approximately one dozen distinct Mu transposons contain highly similar ~215 bp Terminal Inverted Repeats (TIRs) and generate 9-bp Target Site Duplications (TSDs) upon insertion. Using a novel chromosome walking strategy that uses these conserved TIRs as primer binding sites, Mu insertion sites were amplified from Mu stocks and sequenced via the 454 FLX platform.