The experimental flow of mapping insertional mutations in mice with the iPCR method. (a) Sampling. The tail tips of mice are cut for DNA extraction. (b) iPCR procedure. Extracted mouse genomic DNA will go through restriction digestion, self ligation, PCR with PB specific primers to obtain PB flanking genomic DNA fragment. (c) Sequencing. Obtained PB flanking genomic DNA fragment is sequenced with PB specific primer to gather PB flanking genomic DNA sequence. (d) Mapping insertion onto the genome. The genomic sequence is submitted to Ensembl http://www.ensembl.org/Multi/blastview webcite to blast against mouse genome assembly to obtain location of PB insertion. This part is modified according to the blast result shown in Ensembl blastview website. (e) Genotyping (GT) verification. Primer specific to genomic DNA fragment around PB insertion is designed. This primer is paired with a PB specific primer for amplification of a DNA fragment containing both genomic DNA and PB. Success of this amplification means the insertion is GT verified.
Yang et al. BMC Genomics 2009 10(Suppl 3):S7 doi:10.1186/1471-2164-10-S3-S7