. A simple approach is described to ef®ciently amplify DNA sequences¯anking transposon Tn5 insertions. The method involves: (i) digestion with a restriction enzyme that cuts within Tn5; (ii) self-ligation under conditions favouring the production of monomeric circles; (iii) four parallel PCR reactions using primers designed to amplify left or right¯anking sequences, and to distinguish target amplicons from non-speci®c products. This reveals the number of Tn5 insertions and the size of¯anking genomic restriction fragments, without Southern blot analysis. The ampli®ed product contains restriction sites that facilitate cohesive-end cloning. This rapid method is demonstrated using Tn5 and Tn5-Mob tagged DNA sequences involved in albicidin biosynthesis in Xanthomonas albilineans. It is generally applicable for ef®cient recovery of DNA sequences¯anking transposon Tn5 derivatives in insertional mutagenesis studies.
ABSTRACT. The signal transducer and activator of transcription (STAT) genes are responsive to a wide range of cytokines, growth factors, and hormones, and thus control important biological processes. In humans, STAT4 mutations have been identified as genetic markers for rheumatoid arthritis, systemic lupus erythematosus, and primary Sjögren's syndrome, whereas little research has been conducted on bovine STAT4 mutations and their potential effects. Herein, 585 Chinese Holstein cows were used to investigate STAT4 mutations and their effects on milk performance traits. One haplotype block, containing g.95879G>A, g.96013G>C, was identified in intron 20 of the bovine STAT4 gene by restriction fragment length polymorphism-polymerase chain reaction and DNA sequencing. Two single nucleotide polymorphisms were significantly associated with milk yield at 305 days (P < 0.05), and with protein percentage (P < 0.05). Chinese Holstein cows with the haplotype GGGG had higher milk yields at 305 days and lower protein percentages. These results suggest that the 2 single nucleotide polymorphisms of STAT4 could be used as genetic markers for milk performance traits in Chinese Holstein cows.
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