Isolation and sequencing of short cell-surface-bound DNA.
To isolate the DNA fragments bound to cell-surface proteins with high affinity, peptide-DNA complexes were obtained by mild treatment of human endothelial cells with trypsin. After binding of nucleoprotein complexes to BrCN-Sepharose and extensive washing, 7-11 nt DNA fragments were eluted with acidic buffer, labeled with (32)P, and isolated by PAGE. The fragments were ligated to double-stranded oligonucleotide adapters, amplified by PCR, and cloned in pBluescript II KS (+) vector. Sequences of 12 clones containing the insert of cell-surface-bound 7-11-mer DNA fragments were obtained.
Mal'shakova
Vera S
VS
Institute of Chemical Biology and Fundamental Medicine, Siberian Division of the Russian Academy of Sciences, Novosibirsk, Russia. vera_mal@niboch.nsc.ru
Pyshnyi
Dmitry V
DV
Bondar
Alexander A
AA
Vlassov
Valentin V
VV
Laktionov
Pavel P
PP
D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D009698:Nucleoproteins / Q000235:genetics / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D009698:Nucleoproteins; D004247:DNA; D014357:Trypsin