Construction of libraries enriched for sequence repeats and jumping clones, and hybridization selection for region-specific markers.
We describe a simple and rapid method for constructing small-insert genomic libraries highly enriched for dimeric, trimeric, and tetrameric nucleotide repeat motifs. The approach involves use of DNA inserts recovered by PCR amplification of a small-insert sonicated genomic phage library or by a single-primer PCR amplification of Mbo I-digested and adaptor-ligated genomic DNA. The genomic DNA inserts are heat denatured and hybridized to a biotinylated oligonucleotide. The biotinylated hybrids are retained on a Vectrex-avidin matrix and eluted specifically. The eluate is PCR amplified and cloned. More than 90% of the clones in a library enriched for (CA)n microsatellites with this approach contained clones with inserts containing CA repeats. We have also used this protocol for enrichment of (CAG)n and (AGAT)n sequence repeats and for Not I jumping clones. We have used the enriched libraries with an adaptation of the cDNA selection method to enrich for repeat motifs encoded in yeast artificial chromosomes.
Kandpal
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RP
Department of Genetics, Yale University School of Medicine, New Haven, CT 06510.
Kandpal
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Weissman
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SM
D001483:Base Sequence; D002846:Chromatography, Affinity; D002874:Chromosome Mapping; D018244:Chromosomes, Artificial, Yeast; D015698:Genomic Library; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012091:Repetitive Sequences, Nucleic Acid; D014960:X Chromosome
D009838:Oligodeoxyribonucleotides