A rapid isolation of the unknown 5'-flanking sequence of human CENP-B cDNA with polymerase chain reactions.
We rapidly and efficiently isolated the 5'-region of cDNA encoding the N-terminal region of human centromere antigen B (CENP-B) including an ATG methionine codon by polymerase chain reactions (PCR). The unknown 5'-flanking sequence of the cDNA was amplified using an adaptor-sequence ligated to the 5' end as a universal primer sequence. To locate the target fragments, we did an additional PCR with another set of two internal primers using samples of the size-fractionated products as templates, rather than using the conventional hybridization procedure. This approach can further be applied to the analysis of other unknown flanking sequences of cDNA or genomic DNA.
Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Japan.
K
K
Sugimoto
M
M
Himeno
D001324:Autoantigens; D001483:Base Sequence; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004268:DNA-Binding Proteins; D005784:Gene Amplification; D015894:Genome, Human; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D013698:Templates, Genetic
D001324:Autoantigens; C497926:CENPB protein, human; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D004247:DNA