High resolution analysis of the timing of replication of specific DNA sequences during S phase of mammalian cells.

A new method, utilizing selective photodegradation of 5-bromo-deoxyuridine (BUdR)-substituted DNA and flow cytometry, has been developed for analyzing the timing of replication of specific DNA sequences. Chemically synchronized Chinese hamster ovary cells were given a pulse of the deoxythymidine analogue, BUdR, at different times during S phase, and flow sorted according to DNA content, before DNA isolation. Newly-replicated, unifilarly BUdR-substituted DNA was selectively degraded by treatment with 33258 Hoechst plus near UV light followed by S1 nuclease digestion; the resistant DNA was analyzed for its content of 18s and 28s rDNA or dihydrofolate reductase (DHFR) sequences via Southern blot analysis. Both the rDNA and DHFR sequences were found to replicate almost entirely during the first quarter of S phase. The approach described should have general utility for analyzing replication kinetics of specific DNA sequences in mammalian cells

Prenatal diagnosis of Duchenne muscular dystrophy: prospective linkage analysis and retrospective dystrophin cDNA analysis

The accuracy of DNA-based prenatal diagnosis of Duchenne muscular dystrophy (DMD) was determined by study of 174 families. Only 60% of families had a living affected male, and 63% had history of a single affected male. Prenatal diagnosis was declined by 47% of mothers whose DNA studies predicted a carrier risk below 2%, and none have had affected sons. Fetal risk was estimated prospectively by linkage analysis using intragenic and flanking RFLPs and retrospectively using dystrophin cDNA analysis for families whose linkage estimates lacked precision. Diagnostic accuracy was determined by comparing predictions with 40 male pregnancy outcomes. On the basis of linkage analysis, we anticipated 3.2 DMD males and observed 3.0. Retrospective cDNA analysis identified deletions in 2 of these 3 males. The combined use of linkage and cDNA deletion analysis provided a highly accurate method for prenatal diagnosis of DMD

Functional genomic analysis of amniotic fluid cell-free mRNA suggests that oxidative stress is significant in Down syndrome fetuses

To characterize the differences between second trimester Down syndrome (DS) and euploid fetuses, we used Affymetrix microarrays to compare gene expression in uncultured amniotic fluid supernatant samples. Functional pathway analysis highlighted the importance of oxidative stress, ion transport, and G protein signaling in the DS fetuses. Further evidence supporting these results was derived by correlating the observed gene expression patterns to those of small molecule drugs via the Connectivity Map. Our results suggest that there are secondary adverse consequences of DS evident in the second trimester, leading to testable hypotheses about possible antenatal therapy for DS