DIFFERENTIAL REPLICATION TIMING OF X-LINKED GENES MEASURED BY A NOVELMETHOD USING SINGLE-NUCLEOTIDE PRIMER EXTENSION

The ratio of two differentially replicating alleles is not constant du ring S phase. Using this fact, we have developed a method for determin ing allele-specific replication timing for alleles differing by at lea st a single base pair. Unsynchronized cells in tissue culture are firs t sorted into fractions based on DNA content as a measure of position in S phase, DNA is purified from each fraction and used for PCR with p rimers that bracket the allelic difference, amplifying both alleles, T he ratio of alleles in the amplified product is then determined by a s ingle nucleotide primer extension (SNuPE) assay, modified as described [Singer-Sam,J. and Biggs,A.D. (1993) Methods Enzymol., 225, 344-351]. We report here use of this SNuPE-based method to analyze replication timing of two X-linked genes, Pgk-1 and Xist, as well as the autosomal gene Gabra-6. We have found that the two alleles of the Gabra-6 gene replicate synchronously, as expected; similarly, the active allele of the Pgk-1 gene on the active X chromosome (Xa) replicates early relati ve to the silent allele on the inactive X chromosome (Xi), In contrast , the expressed allele of the Xist gene, which is on the Xi, replicate s late relative to the silent allele on the Xa.