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United States Department of Agriculture

Agricultural Research Service

Title: A Small-Insert Bovine Genomic Library Highly Enriched for Microsatellite Repeat Sequences

Authors
item Stone, Roger
item Pulido, J - MILLENNIUM PHARMACEUTICAL
item Duyk, G - MILLENNIUM PHARMACEUTICAL
item Kappes, Steven
item Keele, John
item Beattie, Craig

Submitted to: Mammalian Genome
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 12, 1995
Publication Date: N/A

Interpretive Summary: Microsatellites refer to short stretches of the genome in which two, three, or four of the four bases in DNA are repeated. Examples are CA, GGT, or GATA. The number of times these bases are repeated is highly variable among individuals, thus, microsatellites provide a source of simple genetic markers that can be used to construct linkage maps covering the entire genome. Normally, microsatellites are isolated from libraries of genomic clones containing small segments of DNA and only about 1% of the clones contain microsatellites. We have described an approach to enrich genomic libraries so that about 45% of the clones are positive for microsatellites. This approach provides a much larger pool of microsatellite-containing clones to select from which allows for more stringent discrimination against clones that contain undesirable sequence. This seems to be particularly important in the bovine since 30% of the microsatellites are adjacent to other types of repetitive DNA sequence that hamper the gathering of genetic data. The enrichment approach also makes it possible to isolate microsatellite repeats that occur infrequently in the genome such as the three and four base repeats. In contrast to the human, we found that the three and four base repeats were not a viable source of markers in the bovine. We used the more efficient enrichment approach to add 390 new microsatellite markers to the bovine genetic linkage map. These, along with those previously identified by us and others, will provide in excess of 1,000 markers for the bovine. This marker coverage of the bovine genome provides the basis for mapping genetically complex, economically important traits.

Technical Abstract: A bovine genomic phagemid library was constructed with randomly sheared DNA. Enrichment of this single stranded DNA library with CA or GT primers resulted in 45% positive clones. The fourteen percent of positive clones with (CA GT) >12, and not containing flanking repetitive elements, were sequenced and the efficiency of marker production compared to random M13 bacteriophage libraries. Primer sequences and genotyping information are presented for 390 informative bovine microsatellite markers. The genomic frequency for 11 tri- and tetranucleotide repeats were estimated by hybridization to a lambda genomic library. Only GCT, GGT, and GGAT were estimated to have a frequency of >100 per genome. Enrichment of the phagemid library for these repeats failed to provide a viable source of microsatellite markers in the bovine. Comparison of map interval lengths between 100 markers from the enriched library prepared from randomly sheared DNA and M13 bacteriophage libraries prepared from Mbo1 restriction digests suggested no bias in skeletal genomic coverage based on source of small insert DNA. In conclusion, enrichment of the bovine phagemid library provides a sufficient source of microsatellites so that small repeat lengths and flanking repetitive sequences common in the bovine can be eliminated, resulting in a high percent of informative markers.

Last Modified: 7/28/2014
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