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Map and Link Human Genetic Disorders with SSLP Analysis

hich further defined a critical region of chromosome 9 linked to CHH in this pedigree (not shown). Experiments to identify the CHH gene continue.

Conclusions

Use the CastAway system as a convenient, time-saving alternative to traditional poured polyacrylamide gels for mapping studies. Because linkage mapping relies upon a reproducible resolution of bands, which are only minutely different in size, the CastAway system greatly increases the likelihood of correctly identifying alleles and provides more time to test additional markers.

Methods

Polymorphic markers were analyzed by PCR amplification of genomic DNA in the presence of a-[35S]dATP, followed by analysis on CastAway 4.5% or 6% denaturing polyacrylamide gels. PCR amplifications were performed in 25-l reactions containing 50-ng of genomic DNA; 2 units of Taq DNA polymerase; 2.5 pMol of each primer; 200 M of dCTP, dTTP, and dGTP; 2.5 M dATP; 2 mM MgCl2; 10 mM Tris-HCl pH 8.3; 50 mM KCl; and 2 Ci of a-[35S]dATP. Amplification conditions consisted of an initial incubation of 2 minutes at 94C followed by 35 cycles of 94C for 1 minute, 60C for 1 minute, and 72C for 1 minute followed by a 9-minute incubation at 72C. Oligonucleotide primers were purchased from Research Genetics.

REFERENCES

  1. Weber, J.L. and May, P.E. (1989) Am. J. Hum. Genet. 44: 388-396.

  2. Cottingham Jr., R.W., Idury, R.M., and Schaffer, A.A. (1993) Am. J. Hum. Genet. 53: 252-263.

  3. Schaffer, A.A., et al. (1994) Hum. Hered. 44: 225-237.

  4. Stickler, G.B., et al. (1965) Mayo Clin. Proc. 40: 433-455.

  5. Ahmad, N.N., et al. (1991) Proc. Natl. Acad. Sci. USA 88: 6624-6627.

  6. Richards
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