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Title: USE OF DISC-PCR TO CREATE FRAMEWORK MARKERS

Author
item TROYER, DERYL - KANSAS STATE UNIV.
item Rohrer, Gary
item Alexander, Leeson
item Beattie, Craig

Submitted to: Swine Chromosome 7 Workshop
Publication Type: Proceedings
Publication Acceptance Date: 9/15/1995
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Microsatellites (ms) are especially useful as informative anchors for the integration of physical and genetic maps and are thus ideally suited as "framework markers." However, many porcine ms presently available have been isolated from size-selected, short insert random DNA libraries, so they are not amenable to physical mapping. We have developed a strategy for the direct physical assignment of ms as sequence tagged sites. Direct in situ Single Copy (DISC) PCR is a technique in which PCR reagents are applied directly to metaphase chromosomes on-slide, tightly sealed beneath cover slips, and subjected to PCR in a BioOven III. Biotin-dUTP is incorporated into the amplification product and the signal is detected using a variety of chromogenic methods. We have been using the procedure to assign and orient microsatellite linkage groups, and to estimate chromosome coverage. To date we have physically anchored nearly 20 sequence etagged sites using this technique to porcine chromosomes 1p, 1q, 10p, 10q, 13, and 7. In cases where ms mapped by DISC-PCR are available in large genomic clones, FISH has been used to verify the DISC-PCR assignment. Expressed sequence tags that have been localized include alpha-interferon to porcine chromosome 1 and superoxide dismutase I to bovine chromosome 1. In the latter case, a 1:29 translocation was utilized since banding after the DISC-PCR reaction has been problematic. Recently, however, we have discovered a method of subsequent R banding that will enhance the DISC-PCR procedure by allowing chromosome identification and more accurate signal localization. In the future we plan to use DISC-PCR in conjunction with microdissection, microcloning, and FISH to close marker deficient regions that currently exist in the linkage map.