Screening for overlapping bacterial artificial chromosome clones by PCR analysis with an arbitrary primer.
In this article, we used PCR analysis with arbitrary primers (AP-PCR) to screen for overlapping bacterial artificial chromosome (BAC) clones and assembly of contigs. A rice BAC library with three genome equivalents was used to prepare pooled BAC DNA. Twenty-two arbitrary primers were used to survey the pooled BAC DNAs and individual BAC DNAs. Each primer identified 1-10 loci, and the average was 4.4 loci. There were 1-5 overlapping clones in each locus, and the average was 2.5 clones. A total of 245 BAC clones were identified as overlapping by AP-PCR and the identities were confirmed by DNA-DNA hybridization. The 245 BAC clones were then assembled into 80 contigs and 17 single-clone loci. The results indicated that PCR analysis with arbitrary primers is a powerful tool in screening for overlapping BAC clones with high accuracy and efficiency. The use of AP-PCR analysis should speed up the construction of physical maps of the plant and animal genomes, as well as the rice genome.
Xu
J
J
Plant Breeding, Genetics and Biochemistry, International Rice Research Institute, P.O. Box 933, 1099 Manila, Philippines.
Yang
D
D
Domingo
J
J
Ni
J
J
Huang
N
N
D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D016172:DNA Fingerprinting; D017931:DNA Primers; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D018744:DNA, Plant / Q000737:chemistry; D015723:Gene Library; D016680:Genome, Bacterial; D008969:Molecular Sequence Data; D012275:Oryza; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D004269:DNA, Bacterial; D018744:DNA, Plant