Microsatellites obtained using strand extension: an enrichment protocol.
A new method is described to enrich genomic libraries for clones containing microsatellite repeats. The method involves selection on completed M13 genomic libraries rather than on genomic DNA before library construction. It uses two reactions, in which microsatellite oligonucleotides prime strand extension. The first reaction uses a biotinylated primer allowing vectors with microsatellite-containing inserts to be selected with streptavidin-coated magnetic beads. This reaction may be dependent on the strand displacement activity of the Klenow fragment of DNA Polymerase I. The second strand extension reaction is included to improve the relative transformation efficiency of microsatellite-containing clones. In control experiments starting with 0.7% microsatellite-containing clones, enrichment averaged 99.5%. The method was tested empirically on antechinus and abalone genomic libraries in which enrichment for (CA)n microsatellites was efficient enough that clones could be sequenced without further screening. This protocol is technically straightforward and permits the isolation of a large number of microsatellite markers in less time than is required to execute traditional protocols involving rounds of filter hybridization.
Paetkau
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Department of Zoology, University of Queensland, St. Lucia, Australia.
D017104:Bacteriophage M13 / Q000235:genetics; D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers / Q000235:genetics; D005069:Evaluation Studies as Topic; D015698:Genomic Library; D018895:Microsatellite Repeats
D017931:DNA Primers; D004247:DNA