PMID: 9421536Feb 28, 1998Paper

Differential replication timing of X-linked genes measured by a novel method using single-nucleotide primer extension

Nucleic Acids Research
Z XiongA D Riggs

Abstract

The ratio of two differentially replicating alleles is not constant during S phase. Using this fact, we have developed a method for determining allele-specific replication timing for alleles differing by at least a single base pair. Unsynchronized cells in tissue culture are first 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 primers that bracket the allelic difference, amplifying both alleles. The ratio of alleles in the amplified product is then determined by a single nucleotide primer extension (SNuPE) assay, modified as described [Singer-Sam,J. and Riggs,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 relative to the silent allele on the inactive X chromosome (Xi). In contrast, the expressed allele of the Xist gene, which is on the Xi, replicates late relative to the silent allele on the Xa.

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Citations

Aug 3, 2007·Chromosome Research : an International Journal on the Molecular, Supramolecular and Evolutionary Aspects of Chromosome Biology·N D WilsonE V Volpi
May 12, 2006·Genome Research·Alexander A Gimelbrant, Andrew Chess
Jul 11, 2006·Cytogenetic and Genome Research·G P Holmquist, T Ashley
Apr 24, 2004·Proceedings of the National Academy of Sciences of the United States of America·María Gómez, Neil Brockdorff
Jan 26, 2005·The Journal of Cell Biology·Joost GribnauRudolf Jaenisch
Nov 4, 2004·The Journal of Biological Chemistry·Alexander A GimelbrantAndrew Chess

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