The overall aim of this research is to develop a faster, more convenient, DNA diagnostic test which can simultaneously detect many mutant alleles at many different genetic loci. The specific goal of this proposal is to build and validate such a multiplex DNA diagnostic test for the 15 most commonly occurring mutant alleles of the CFTR gene, the gene which is responsible for Cystic Fibrosis in man. The test will be capable of detecting well over 90% of CF mutations and would be useful in population screening as well as for diagnosis of CF mutations in members of families known to segregate mutant alleles. The test utilizes allele-specific primers which also carry sequence-specific tails. Following allele- specific PCR amplification (ASPCR), the products are captured by hybridization to complementary sequences of the tails which are fixed to discrete spots on a membrne. Multiple ASPCRs can be performed in the same reaction vessel, and the multiple ASPCR products can be pooled for hybridization to a single membrane. Detection is by non-radioactive methods using alkaline phosphatase-labeled oligonucleotides or streptavidin-alkaline phosphatase conjugates. The convenience, speed, and virtually limitless potential for multiplexing make this new test superior to and less expensive than currently available DNA diagnostic tests. GRANT-R01HD30587 This project is a longitudinal study of 150 children who will be studied from the first through fifth grades. At the beginning of the study, all children will be enrolled in formal after-school program. Children will be followed over a five-year period as come remain in the formal programs, and others move to informal adult supervision and to self-care. Measures of after-school care include (a) type of care-formal after school programs, self-care, and informal adult supervision, (b) curriculum focus within the formal programs--academic, enrichment, recreational, and (c) quality of the after-school experiences. Child outcomes being studied in relation to these after-school experiences include academic and conduct grades, achievement test scores, work habits, self-esteem, depression, behavior problems, peer relationships, and loneliness. The project has six major goals: (1) to use observations and interviews to ascertain the ways in which children's activities and experiences during the after-school hours vary as a function of type, quality, and curricular focus, (2) to examine the concurrent, lagged, and cumulative impact of variations in type of after- school arrangements over time on children's social, emotional, and academic development, (3) to examine the impact of variations in quality of after-school arrangements over time on children's development, in terms of concurrent, lagged, and cumulative effects, (4) to determine, for those children who attend formal after-school programs, the impact of variations in curricular focus on children's development, (5) to ascertain whether family demographic and psychological characteristics are associated with the use of different after-school arrangements, and (6) to determine if the effects of after-school arrangements are moderated by family or child characteristics.
