Proceedings of the National Academy of Sciences · 1989 · 578 citations · 28 references
Life SciencesGeneticsMolecular GeneticsCytogeneticsGenome EngineeringGenomicsChromosome MappingGene MappingMolecular Cloning WorkflowsPolymerase Chain ReactionYeast Artificial ChromosomesDna ProbesPolymerase Chain Reaction AnalysisSomatic Cell Hybrid Mapping
Current human genome mapping focuses on individual chromosomes or smaller regions and often uses somatic cell hybrids. The study applies PCR to directly amplify human DNA from hybrid cells using primers targeting the human Alu repeat element. PCR with Alu primers amplifies human DNA fragments from hybrid cells and cloned DNA, enabling localization of large X chromosome fragments in yeast artificial chromosomes and preparation of DNA probes from cloned human sequences. Alu-directed PCR amplified a human HPRT fragment, identified the involved Alu repeats, mapped large X chromosome fragments in yeast artificial chromosomes, and proved the method allows rapid gene mapping and simple isolation of specific chromosomal regions.
Current efforts to map the human genome are focused on individual chromosomes or smaller regions and frequently rely on the use of somatic cell hybrids. We report the application of the polymerase chain reaction to direct amplification of human DNA from hybrid cells containing regions of the human genome in rodent cell backgrounds using primers directed to the human Alu repeat element. We demonstrate Alu-directed amplification of a fragment of the human HPRT gene from both hybrid cell and cloned DNA and identify through sequence analysis the Alu repeats involved in this amplification. We also demonstrate the application of this technique to identify the chromosomal locations of large fragments of the human X chromosome cloned in a yeast artificial chromosome and the general applicability of the method to the preparation of DNA probes from cloned human sequences. The technique allows rapid gene mapping and provides a simple method for the isolation and analysis of specific chromosomal regions.
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