RESUMO
An HPLC approach for purification and sequencing of double-stranded DNA obtained directly from a PCR is described. This simple and reliable procedure has several advantages; the DNA fragment is rapidly eluted (less than 7 minutes), requires no organic cleanup, produces several hundred bases of sequence and is sensitive enough to obtain DNA sequence from a single 100-microliters PCR. This method is demonstrated by sequencing tumor necrosis factor alpha (TNF alpha) gene amplified from mouse tail DNA.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , DNA/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Animais , Sequência de Bases , Camundongos , Dados de Sequência Molecular , Polidesoxirribonucleotídeos , Fatores de TempoRESUMO
We have cloned 60 kilobases of overlapping human genomic DNA comprising the complete coding sequence of the biosynthetic enzyme beta-1,4-galactosyltransferase (GalTase). The human locus spans greater than 50 kb of genomic DNA and shows an exon structure similar to the mouse gene. However, contrary to the mouse and bovine systems, which yield two distinct transcripts, RNA blotting and RNase protection analysis of human mRNA derived from HeLa cells reveal only a single transcript, corresponding to the "short" form of the mouse and bovine transcripts. Furthermore, Northern analysis on a panel of human cell lines of varying tissue origin and morphology shows GalTase expression levels to be highly variable, consistent with the notion that GalTase expression and consequent cell-specific differences in galactosylation are at least partially regulated at the transcriptional level.
