We have generated a human liver cDNA library and have developed a protocol for identifying cDNA clones within the library. The screening is based on pooling 5 to 10 cDNA clones on DBM filters and using plasmid selection of human or baboon poly(A)+RNA followed by immunoprecipitation and analysis on SDS polyacrylamide gels. The screening utilizes pooling of antibodies as well as cDNA clones so that a single lane in the gel analysis can represent 20 to 100 tests. We have identified a clone for human Alpha1-antitrysin and propose to use this clone to identify restriction fragment length polymorphisms to carry out prenatal diagnosis of the deficiency state. We propose to generate an improved, larger unfractionated cDNA library as well as 10 to 12 sublibraries based on fractionation of the poly(A)+RNA on a sucrose gradient. We propose to identify new cDNA clones for gene products of 0.1% or greater abundance. Currently screening is utilizing antibodies against argininosuccinate lyase, arginase, numerous apolipoproteins and numerous plasma proteins. We will focus specifically on attempts to clone cDNAs for carbamylphosphate synthetase, ornithine transcarbamylase and glucose-6-phosphatase. We will make the libraries available to other investigators. We will use DNAs for medical and biological applications such as molecular analysis of human diseases, prenatal diagnosis and gene mapping.
| O'Brien, W E; McInnes, R; Kalumuck, K et al. (1986) Cloning and sequence analysis of cDNA for human argininosuccinate lyase. Proc Natl Acad Sci U S A 83:7211-5 |
| Fearon, E R; Mallonee, R L; Phillips 3rd, J A et al. (1985) Genetic analysis of carbamyl phosphate synthetase I deficiency. Hum Genet 70:207-10 |
