Identification of a Gene That Causes Primary Open Angle Glaucoma
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Kahn H. A., Moorhead H. B., U.S. Public Health Serv. Publ. NIH 73-4271973120.
Tielsch J. M., Transactions of the New Orleans Academy of Ophthalmology, Ball S. F., Franklin R. M.KuglerAmsterdam19936168.
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Sheffield V. C. Stone E. M.unpublished data.
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Nguyen T. D. Polansky J. R. Huang W.unpublished data; International patent application PCT/US95/14024 (1996).
YAC-STS content analysis was performed by PCR amplification of each STS from DNA prepared from individual YAC clones. PCR products were electrophoresed on 2% agarose gels and stained with ethidium bromide. The presence or absence of amplification was scored and this data used to deduce the minimum tiling path of the chromosome 1q glaucoma interval. Radiation hybrid mapping was performed with the Genebridge 4 radiation hybrid panel available from Research Genetics. DNA from each radiation hybrid clone was amplified in duplicate with STS primers. PCR products were electrophoresed on 2% agarose gels and visualized with ethidium bromide. The gels were scored for the presence of amplified product. Radiation hybrid maps based on this data were constructed with the computer programs RH2PT, RHMAXLIK, and RHMINBRK [ Lange K. , Boehnke M. , Cox D. R. , Lunetta K. L., Genome Res. 5, 136 (1995)].
A 12.5-ng sample of each patient's DNA was used for the template in an 8.35-μl PCR mixture containing 1.25 μl of 10X buffer (100 mM tris-HCL pH 8.3 500 mM KCl 15 mM MgCl 2 ); the deoxynucleotides dCTP dATP dGTP and dTTP (300 μM for each); 1 pmol of each primer; and 0.25 units of Taq polymerase (Boehringer Mannheim). Samples were denatured for 5 min at 94°C and incubated for 35 cycles under the following conditions: 94°C for 30 s 55°C for 30 s and 72°C for 30 s in a DNA thermocycler (Omnigene). After amplification 5 μl of stop solution (95% formamide 10 mM NaOH 0.05% bromophenol blue 0.05% xylene cyanol) was added to each sample. Amplification products were denatured for 3 min at 94°C and electrophoresed on 6% polyacrylamide-5% glycerol gels at 25 W for about 3 hours. After electrophoresis gels were stained with silver nitrate (24).
PCR products were sequenced with fluorescent dideoxynucleotides on an Applied Biosystems (ABI) model 373 automated sequencer. All mutations were recognized by the approximately equal peak intensity of two fluorescent dyes at the mutant base. Mutations were confirmed by cloning the PCR product into pT7Blue T vector (Novagen Madison WI) and sequencing the cloned product with ABI fluorescent dye primer chemistry. Multiple clones were sequenced to confirm the presence of both mutant and normal clones. All sequencing was performed bidirectionally.
Polansky J. R., Basic Aspects of Glaucoma Research III, Lutjen-Drecoll E.SchattauerStuttgart, New York1993307318T. D. Nguyen et al., in ibid., pp. 331–343.
Mao L. K. , Stewart W. C. , Shields M. B., ibid. 111, 51 (1991).
This paper is dedicated to the memory of Frederick C. Blodi M.D. (1917–1996). We thank G. Beck N. Butler D. Crouch R. Hockey T. Love T. Rohklina L. Streb C. Taylor C. Wiles and K. Vandenburgh for their technical assistance. Supported in part by the Carver Charitable Trust NIH grants EY10564 EY08905 EY02477 and EY02162 and an unrestricted grant from Research to Prevent Blindness New York NY.