Rapid communication: genetic linkage and physical mapping of the porcine phospholipid transfer protein (PLTP) gene.

Source and Description of Primers. Primers for the Phospholipid Transfer Protein gene (PLTP) were designed from homologous regions of published human and mouse PLTP sequences (GenBank accession number NM_006227 and NM_011125, respectively). The primers were used to amplify a 1,368-bp fragment of the porcine PLTP gene spanning an intron between exons 4 and 5. Porcine PLTP sequences were sequenced from animals of the Yorkshire and Meishan breeds (GenBank accession number AF295932) and showed 93% (86/94) nucleotide identity to the corresponding human PLTP exon sequence. Additionally, a pig-specific forward primer was designed for use in PCR-RFLP analysis and in the pig/rodent somatic cell hybrids to map the PLTP gene by genetic linkage and physical analysis, respectively (see below). Primer Sequences. Primers derived from human and murine sequences were as follows: forward PF1: 5′-AC GGATGAAAGTGTCCAATGT-3′; reverse PR1: 5′-GTT GAGGAGGAAGCGCATC-3′. The pig-specific forward primer PF2 was 5′-TGAAGGGTAGGACAAGACAG-3′. An internal primer (PS1) used for sequencing was 5′ACTGTCTTGTCCTACCCATC-3′. Method of Detection. The PCR amplification was performed using 25 ng of porcine genomic DNA, 1× PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.15 M of each primer (PF2 and PR1), and 0.5 U Taq polymerase (Promega, Madison, WI) in a 10L reaction volume. The PCR profile included 3 min at 94°C, 40 cycles of 45 s at 94°C, 45 s at 62°C, 1 min at 72°C, and a final 5-min extension at 72°C in an MJ Research model PTC-200 (Watertown, MA). The 505-bp PCR product was di-