Submitted to: Biotechniques
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 5, 2003
Publication Date: November 1, 2003
Citation: Waldbieser, G.C., Quiniou, S.M.A., Karsi, A. 2003. Rapid Development of Gene-Tagged Microsatellite Markers from BAC Clones Using Anchored TAA-Repeat Primers. Biotechniques. 35:976-979. Interpretive Summary: Identification of DNA sequence variations in individual genes allows researchers to correlate specific variants with performance characteristics such as growth rate, disease resistance, and carcass quality and yield. Researchers at the USDA, ARS, Catfish Genetics Research Unit have developed a novel approach to identification of gene-linked variations in DNA sequences by direct sequencing of large insert DNA clones with short tandem repeat-anchored primers (STRAPs). STRAP sequencing permits rapid development of polymorphic markers linked to known genes and the technique can be used in many eukaryotic species. This will permit researchers to add genes conserved between species to genetic linkage maps and screen populations for variation within or near genes that control important traits.
Technical Abstract: A technique was developed to improve the efficiency of producing TAA-repeat microsatellite markers linked to interspecific conserved genes. Template DNA was prepared from cultures derived from single BAC colonies using a simple alkaline lysis miniprep. Presence of conserved genes in each BAC clone was verified by sequencing with gene-specific primers. The BAC templates were directly sequenced using primers consisting of TAA repeats with one or two unique 3' terminal bases. These primers were termed STRAPs for 'Short Tandem Repeat-Anchored Primers'. At least one STRAP provided sufficient 3' flanking sequence from each clone for design of a BAC-specific primer. The BAC-specific primer was used to sequence back through the tandem repeat and obtain 5' flanking sequence, and a second BAC-specific primer was designed for microsatellite genotype analysis. This technique quickly provided microsatellite markers with an average of 15 tandem repeats for the BAC clones tested. Identification of polymorphic microsatellite loci in these clones permits identification of alleles linked to candidate genes, placement of conserved genes on genetic linkage maps, and integration of linkage and physical maps.