Rapid isolation of a rice waxy sequence: a simple PCR method for the analysis of recombinant plasmids from intact Escherichia coli cells.
Polymerase chain reaction (PCR) is an efficient method for obtaining a desired nt sequence if both required primers can hybridize to the target DNA molecule specifically. A rapid and simple PCR-based method for analyzing plasmids using intact cells was established. An attempt to target a rice waxy sequence by PCR using homologous primers was also carried out. In three cases, specific fragments were amplified and their nucleotide sequences were determined. However, the cloned rice waxy gene contained base substitution mutations. The cumulative frequency of mutation after 30 polymerization cycles was estimated to be one in 500 bp.
Shimada
H
H
Mitsui Plant Biotechnology Research Institute, Tsukuba, Japan.
Tada
Y
Y
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004274:DNA, Recombinant / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D001467:Hordeum / Q000235:genetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D012275:Oryza / Q000235:genetics*; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D012689:Sequence Homology, Nucleic Acid; D003313:Zea mays / Q000235:genetics
D004274:DNA, Recombinant; D015345:Oligonucleotide Probes