Comparative genomics by capture PCR.
There is increasing demand for efficient methods to relate genomic information from model organisms to other species of interest. Comparative genetic analyses are particularly valuable to identify functionally important sequence features on the basis of their evolutionary conservation. We demonstrate here how a single segment of just 32 or less conserved coding nucleotide positions can be used to isolate homologous gene sequences from large numbers of species using a single-sided PCR technique. The method was used to isolate and determine the 3'-untranslated sequence of the somatostatin gene from vertebrate species ranging from human to hagfish. Two sequence motifs centered an average 40-145 nucleotides downstream of the translational stop codon have remained conserved for up to 350 million years. One of the conserved tetrapod segments was used to select a primer for amplification of so-called comparative anchor tagged sequences (CATS) in regular PCR, and shown to amplify homologous sequences from DNA samples from 30 out of 33 tetrapods. In conclusion, we present a useful procedure to reveal functionally relevant sequence elements, and to select primers for amplification of homologous sequences from a wide range of species.
Lagerström-Fermér
Maria
M
Department of Medical Sciences, Unit of Molecular Medicine, University Hospital, Uppsala, Sweden. Maria. Lagerstrom@medsci.uu.se
Larhammar
Dan
D
Johnsen
Elsy
E
Landegren
Ulf
U
D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D023281:Genomics / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D013004:Somatostatin / Q000235:genetics
D020413:3' Untranslated Regions; D013004:Somatostatin