Haplotype Analysis Involving a Novel Polymorphism in Bovine Alpha-LactaIbumin 5' Flanking Region

摘要

DNA was recovered from 5 ml of blood samples from dairy cattle (n = 353) with a commercial kit (Qiagen, Valencia CA) and stored at 4 deg. C until characterized for polymorphisms. The 429-bp fragment was produced ini- tially with primers described by Voelker et al. (1997). Reaction conditions were an initial 2 min at 94 deg. C, fol- lowed by 30 cycles of 94 for 30 s and 57 deg. C for 30 s. A 72 deg. C extension phase was not used during cycles, but the final cycle was followed by a 30-s extension phase at 72"C. Final MgCl_2 concentration was 1.1 InM. PCR products from animals homozygous for alternate alleles [as determined by single-strand conformation polymor- phism (SSCP) technique] were cloned into a vector us- ing a commercial kit (TOPO Cloning Kit, InVitroGen, Carlsbad CA). Plasmid DNA was extracted from posi- tive clones with a commercial kit (Qiagen, Valencia CA). Sequencing reactions were performed, at least twice in both directions, using BigDye reagents and procedure and analyzed on an ABI377 automated sequencer (PE Applied Biosystems, Foster City CA).