The Genetic mapping of single-gene phenotypes by linkage analysis with PCR-amplified microsatillites from pooled DNA.

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Testing for linkage with recently discovered mouse mutations is tedious and time-consuming, as it can take up to 1200 PCR reactions to discover the approximate location of one such gene. Two mouse hybrid crosses (a backcross and an intercross) were analyzed for the feasibility of using pooled DNA from mice with like phenotypes to detect linkage with microsatellite markers. By using pooled DNA, the number of reactions needed could be cut down to one-tenth of the original estimate. For some microsatellite markers, linkage was easily detectable with pooled DNA up to approximately 20 cM or 20% recombination. Some markers worked well and some amplified on ly one allele, showing the importance of using parental controls in all instances. It would be feasible to use three or four equally interspersed markers for each chromosome when mapping, as this also cuts down on the number of reactions. In the intercross investigation, one optimum marker near the centromere was found: D19Mit14. For the backcross investigation, three equally interspersed markers were chosen as optimum for linkage tests: D18Mit30, D 18Mit14, and D18Mit7. Overall, pooling DNA appears to be a feasible protocol for improving the efficiency of mapping genetic mutations. However, in order for it to be put to use, optimum markers on every chromosome need to be tested and chosen.


August 13, 1993

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