Genome mapping by arbitrary amplification of yeast artificial chromosomes.
Several methods have been described for using the polymerase chain reaction (PCR) to isolate fragments of DNA for genome mapping. We have developed an approach for isolating discrete fragments by amplifying DNA with single oligonucleotides (10-mers) with arbitrarily selected sequences. The method is rapid and technically simple. We isolated fragments from a contig of three yeast artificial chromosomes (YACs) from the human Xq28 chromosomal region. We purified YACs yWXD37, yWXD348, and yWXD705 from a preparative pulsed field gel. Amplifications of each YAC were performed with single 10-mers as the PCR primers and the products were visualized on agarose gels. These fragments have been successfully used as hybridization probes against Southern blots containing the YACs and against blots containing human genomic DNA and somatic cell hybrids containing Xq28 as their only human constituent. The results have been concordant with the known order of the YACs. We have also successfully combined 10-mers with primers derived from vector arm sequences to isolate YAC ends. We discuss several uses of this method in comparative mapping and in filling in gaps in physical and genetic maps.
Di Rienzo
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Department of Psychiatry, University of California, San Francisco 94143.
Peterson
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A
Das
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S
Freimer
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NB
D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods*; D015825:Chromosomes, Fungal; D004247:DNA / Q000235:genetics; D015342:DNA Probes; D005069:Evaluation Studies as Topic; D005784:Gene Amplification; D015723:Gene Library; D005822:Genetic Vectors; D015894:Genome, Human; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods; D014960:X Chromosome
D015342:DNA Probes; D004247:DNA