A simple and rapid nucleotide sequencing strategy and its application in analyzing a rice histone 3 gene.
An improved rapid method for sequencing a target DNA is described. A new plasmid, pAA-PZ1, which contains the origin of replication from phage M13 and a portion of the Tn9 transposon was constructed. A long fragment of target DNA cloned into this vector is progressively shortened in vivo from one end by transposon-mediated deletions. The plasmids carrying different lengths of target DNA are then made into single-stranded DNA in the same host upon infection with an M13 phage and their sequence is determined using the dideoxynucleotide chain-termination method. This method bypasses the in vitro enzymatic manipulations for progressive deletions and requires no subcloning. Using this strategy, we sequenced 1.3 kb of rice DNA containing a histone 3 gene within three weeks.
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D004262:DNA Restriction Enzymes; D005796:Genes; D005818:Genetic Engineering / Q000379:methods; D006657:Histones / Q000235:genetics*; D012275:Oryza / Q000235:genetics; D010944:Plants / Q000235:genetics*; D010957:Plasmids
D006657:Histones; D004247:DNA; D004262:DNA Restriction Enzymes