Standard mapping protocol
To map new mouse mutations we use CAST/Ei, an inbred strain of Mus musculus castaneus, as our standard linkage testing strain. In some cases, because of breeding difficulties or reduced phenotypic penetrance, we use other strains. An intercross of F1 hybrids is usually used to analyze linkage of recessive mutations and a backcross to analyze linkage of dominant mutations. Our goal is to produce enough informative mice from each mapping cross to test the recombinational products from at least 100 meioses.
DNA is extracted from the frozen tail tips of mutant (homozygous) F2 mice or backcross progeny by a standard hot sodium hydroxide and Tris (Hot SHOT) procedure (Truett, et al., 2000) or from spleens using standard phenol extraction methods.
Polymerase chain reaction
PCR primer pairs (MapPairs, from Research Genetics, Huntsville, Ala., or from Integrated DNA Technologies, Coralville, Ia.) are used to type MIT microsatellite markers positioned throughout the genome. PCR reactions contain 20 ng genomic DNA in 10 ul containing 50 mM KCL, 10 mM Tris-HCL (pH 9.0 at 250C), and 0.01% Triton-X-100, 2.25 mM MgCl2, 100 nM of each primer (forward and reverse), 100 uM of each of four deoxyribonucleoside triphosphates, and 0.5 Units of Taq DNA polymerase (Amplitaq from Applied Biosystems #N808-0145). Amplification consists of one cycle of denaturation at 940C for 3 minutes followed by 30 cycles, each consisting of 940C for 15 sec. denaturation, 550C for 2 minutes of annealing, and 720C for 2 minutes of extension. After the 30 cycles, the final product is extended for 7 minutes at 720C. The PCR products are run on 2.5% Metaphor agarose gels or 6% polyacrylamide (non-denaturing) gels. The gels are then stained with ethidium bromide, destained with distilled water, visualized on a UV light table, and photographed.
Pooled DNA Method (Taylor et al 1994)
In the case of an intercross, a pool of DNA prepared from 25-30 mutant F2 mice is compared with DNA from F1 hybrids. In the case of a backcross, a DNA pool from 25-30 mutant N2 mice is compared with a pool from 25-30 unaffected N2 mice. These DNA samples are typed by PCR for MIT markers located throughout the genome. For linked markers, the mutant strain allele will predominate in the DNA pool from mutant mice compared with controls. When a particular marker indicates linkage by analysis of the pooled sample, individual DNA samples are typed with that marker and additional markers in the same region to confirm linkage. Once a linkage is confirmed, additional DNAs from individual mice are typed to obtain a finer map position.
Gene order and recombination frequencies are calculated with the Map Manager computer program (Manley1993, 2001), a MacIntosh program for storage and analysis of genotyping data.
Manly KF (1993) A MacIntosh program for storage and analysis of experimental mapping data. Mamm Genome 4: 303-313.
Manly KF, Cudmore RH Jr, and Meer JM (2001) Map Manager QTX, cross-platform software for genetic mapping. Mamm Genome12: 930-932.
Taylor BA, Navin A, and Phillips SJ (1994) PCR-amplification of simple sequence repeat variants from pooled DNA samples for rapidly mapping new mutations of the mouse. Genomics 21: 626-32.
Truett GE, Heeger P, Mynatt RL, Truett AA, Walker JA, and Warman ML(2000) Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and Tris (HotSHOT). Biotechniques 29:52-54