Construction of small-insert genomic DNA libraries highly enriched for microsatellite repeat sequences.
We describe an efficient method for the construction of small-insert genomic libraries enriched for highly polymorphic, simple sequence repeats. With this approach, libraries in which 40-50% of the members contain (CA)n repeats are produced, representing an approximately 50-fold enrichment over conventional small-insert genomic DNA libraries. Briefly, a genomic library with an average insert size of less than 500 base pairs was constructed in a phagemid vector. Amplification of this library in a dut ung strain of Escherichia coli allowed the recovery of the library as closed circular single-stranded DNA with uracil frequently incorporated in place of thymine. This DNA was used as a template for second-strand DNA synthesis, primed with (CA)n or (TG)n oligonucleotides, at elevated temperatures by a thermostable DNA polymerase. Transformation of this mixture into wild-type E. coli strains resulted in the recovery of primer-extended products as a consequence of the strong genetic selection against single-stranded uracil-containing DNA molecules. In this manner, a library highly enriched for the targeted microsatellite-containing clones was recovered. This approach is widely applicable and can be used to generate marker-selected libraries bearing any simple sequence repeat from cDNAs, whole genomes, single chromosomes, or more restricted chromosomal regions of interest.
Ostrander
E A
EA
Department of Molecular and Cellular Biology, University of California, Berkeley 94720.
Jong
P M
PM
Rine
J
J
Duyk
G
G
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002999:Clone Cells; D004247:DNA / Q000235:genetics*; D004251:DNA Transposable Elements; D004276:DNA, Satellite / Q000235:genetics*; D004285:Dogs; D015698:Genomic Library; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid
D004251:DNA Transposable Elements; D004276:DNA, Satellite; D009838:Oligodeoxyribonucleotides; D004247:DNA