International Mammalian Genome Society

The 14th International Mouse Genome Conference (2000)

H13. Routine Flanking Sequence Tag Determination for Gene Trap Vector Insertions in Mouse by TAIL-PCR

Robert F. Whittier1, Tomoko Ohira1, Masanori Gotoh1, Kimi Araki2 and Ken-ichi Yamamura2
1Amersham Pharmacia Biotech K.K., R&D, Hyakunincho 3-25-1, Shinjuku-ku, Tokyo 169-0073, Japan and 2Institute of Molecular Embryology and Genetics, Kumamoto University, Kuhonji 4-24-1, Kumamoto 862-0976, Japan.

Mutagenesis with gene trap insertion vectors constitutes a powerful means of pursuing functional genomics, enabling systematic association of mutant knockout phenotypes with their respective genes. Unlike more easily generated mutants, the presence of a known unique sequence in insertion mutants enables identification of the affected gene through recovery of flanking cDNA or genomic sequence tags (FSTs). However, once the mutants are in hand, what is the most efficient method for determining the FSTs? While plasmid rescue and inverse PCR are widely known and practiced by dint of their early development, the manipulations are quite involved and therefore recalcitrant to automation and high throughput processing. We present here our success in adapting thermal asymmetric interlaced (TAIL-) PCR for isolation of mouse genomic FSTs, a method previously utilized in the much smaller Arabidopsis genome. In this method serial reactions are carried out with nested sequence-specific primers and a shorter arbitrary degenerate primer that is adapted to prime synthesis from within the unknown flanking sequence. Interspersion of high with lower stringency cycles allows logarithmic amplification of target sequences while suppressing the amplification of undesired nontarget sequences primed by the arbitrary primer alone. The major advantage of this approach is that all reactions through sequencing can be carried out by simple dilution of each reaction into the next, making the method amenable to automation and parallel processing of samples.

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