Molecular cloning of large alternative transcripts based on comparative phylogenetic analysis and exploration of an EST database

In animals, a gene may be 50 kb or over and contain multiple alternative transcripts with sequences that are not experimentally validated. Under these special circumstances, PCR-based cloning may become very difficult. Here a simple cloning strategy is described using the mNLRC5 gene as an example. We performed comparative phylogenetic analysis between murine and human NLR protein families to anchor the translation start codon, searched an EST database with the 3′ end of the genomic DNA sequence to obtain ESTs from the farthest 3′ end of the gene, and isolated the full-length CDS of the mNLRC5 of about 6 kb through conventional RT-PCR and 3′ RACE.