Chromosome-specific microsatellite multiplex sets for linkage studies in the domestic dog

doi:10.1016/j.ygeno.2004.06.006

Genomics

Volume 84, Issue 3, September 2004, Pages 550-554

https://doi.org/10.1016/j.ygeno.2004.06.006 Get rights and content

Abstract

To expedite linkage studies and positional cloning efforts in the dog, Minimal Screening Set 2 (MSS-2) of 327 canine microsatellite markers has been multiplexed into chromosome-specific panels. MSS-2 provides 9 Mb coverage of the canine genome with no gaps larger than 17.1 Mb and is the most recent and comprehensive set of microsatellites available for whole-genome scans. Markers were labeled with fluorescent dyes based on locations and expected product sizes to facilitate the multiplexing of a maximum number of markers for each chromosome. All markers are amplified using a single thermal cycling program and PCR mix and are optimized for resolution on an ABI 3100 genetic analyzer. Sixty-nine chromosome-specific panels were created by coamplification of a maximum number of markers and subsequent coloading of the remaining markers.

Section snippets

Results

Three hundred sixteen microsatellite markers from MSS-2 are resolved in 69 chromosome-specific panels (Table 1), providing an average of 1.73 multiplex sets per chromosome. Two hundred ninety-six markers can be coamplified within the chromosome panels. The remaining 20 markers are amplified individually and coloaded into designated panels for resolution in a single capillary. Eight markers are coamplified in pairs and are then coloaded with the appropriate panel. Three markers, FH3245,

Discussion

The most comprehensive screening set currently defined for linkage studies in the dog is MSS-2, which offers 9 Mb coverage and highly polymorphic markers, including 64 markers from the MSS-1. To enhance the utility of MSS-2, we have developed chromosome-specific multiplex sets, which expedite whole genome scans in the dog and have the potential to exclude candidate genes on a given chromosome. Collection of data by chromosome also allows for statistical analysis for individual chromosomes to be

Materials and methods

Primer pairs were synthesized by Applied Biosystems (PE Biosystems, Foster City, CA, USA) and forward primers were labeled with one of four fluorescent dyes: 6FAM, NED, PET, or VIC. Previously multiplexed microsatellites [6] that were labeled with 6FAM were not relabeled, and those labeled with TET and HEX were relabeled with VIC and NED, respectively, to retain their original dye colors. Dye types for new markers were chosen for even distribution across each chromosome and size range. Primers

Acknowledgment

E.A.O., F.G., and K.E.M. gratefully acknowledge support from the American Kennel Club–Canine Health Foundation.

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¹: These authors contributed equally to this work.

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Chromosome-specific microsatellite multiplex sets for linkage studies in the domestic dog

Abstract

Section snippets

Results

Discussion

Materials and methods

Acknowledgment

Trends Genet.

J. Biochem. Biophys. Methods

Genomics

Unleashing the canine genome

Genome Res.