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Screening for overlapping bacterial artificial chromosome clones by PCR analysis with an arbitrary primer.

ABSTRACT: 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.

SUBMITTER: Xu J

PROVIDER: S-EPMC20435 | biostudies-literature | 1998 May

REPOSITORIES: biostudies-literature

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Screening for overlapping bacterial artificial chromosome clones by PCR analysis with an arbitrary primer.

Xu J J Yang D D Domingo J J Ni J J Huang N N

Proceedings of the National Academy of Sciences of the United States of America 19980501 10

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 ...[more]

PMID: 9576940

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Project description:BackgroundCurrent techniques of screening bacterial artificial chromosome (BAC) libraries for molecular markers during the construction of physical maps are slow, laborious and often assign multiple BAC contigs to a single locus on a genetic map. These limitations are the principal impediment in the construction of physical maps of large eukaryotic genomes. It is hypothesized that this impediment can be overcome by screening multidimensional pools of BAC clones using the highly parallel Illumina GoldenGate assay.ResultsTo test the efficacy of the Golden Gate assay in BAC library screening, multidimensional pools involving 302976 Aegilops tauschii BAC clones were genotyped for the presence/absence of specific gene sequences with multiplexed Illumina GoldenGate oligonucleotide assays previously used to place single nucleotide polymorphisms on an Ae. tauschii genetic map. Of 1384 allele-informative oligonucleotide assays, 87.6% successfully clustered BAC pools into those positive for a BAC clone harboring a specific gene locus and those negative for it. The location of the positive BAC clones within contigs assembled from 199190 fingerprinted Ae. tauschii BAC clones was used to evaluate the precision of anchoring of BAC clones and contigs on the Ae. tauschii genetic map. For 41 (95%) assays, positive BAC clones were neighbors in single contigs. Those contigs could be unequivocally assigned to loci on the genetic map. For two (5%) assays, positive clones were in two different contigs and the relationships of these contigs to loci on the Ae. tauschii genetic map were equivocal. Screening of BAC libraries with a simple five-dimensional BAC pooling strategy was evaluated and shown to allow direct detection of positive BAC clones without the need for manual deconvolution of BAC clone pools.ConclusionThe highly parallel Illumina oligonucleotide assay is shown here to be an efficient tool for screening BAC libraries and a strategy for high-throughput anchoring of BAC contigs on genetic maps during the construction of physical maps of eukaryotic genomes. In most cases, screening of BAC libraries with Illumina oligonucleotide assays results in the unequivocal relationship of BAC clones with loci on the genetic map.

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Screening for overlapping bacterial artificial chromosome clones by PCR analysis with an arbitrary primer.

Publications

Screening for overlapping bacterial artificial chromosome clones by PCR analysis with an arbitrary primer.

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