Isolation of candidate genes for macular degeneration using an improved solid-phase subtractive cloning technique.
An improved solid-phase subtraction procedure was developed to generate a readily amplifiable library of short cDNA fragments highly enriched in the macula (target) versus the peripheral region (driver) of the monkey neural retina. The generated clones were sequenced and 63 were analyzed by northern blotting using total RNA from the monkey macula and peripheral retina. The results indicate that 32% are highly enriched in macula, 36% are below the limits of detection and 32% are not enriched. No clones were found which were enriched in the peripheral retina. Our technique is therefore successful in identifying novel cDNAs enriched in the macula area of the neural retina that may represent potential candidate genes for hereditary ocular diseases. It should thus be useful in other situations where subtle differences in expression between cell types or tissue areas need to be analyzed.
Schoen
T J
TJ
Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Mazuruk
K
K
Chader
G J
GJ
Rodriguez
I R
IR
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002874:Chromosome Mapping; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D018076:DNA, Complementary; D015723:Gene Library; D006801:Humans; D008253:Macaca mulatta; D008268:Macular Degeneration / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012160:Retina / Q000378:metabolism*
D017931:DNA Primers; D018076:DNA, Complementary