Differential replication timing of X-linked genes measured by a novel method using single-nucleotide primer extension.
Nucleic Acids Res
; 26(2): 684-6, 1998 Jan 15.
Article
em En
| MEDLINE
| ID: mdl-9421536
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Cromossomo X
/
Primers do DNA
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RNA não Traduzido
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Replicação do DNA
Limite:
Animals
Idioma:
En
Revista:
Nucleic Acids Res
Ano de publicação:
1998
Tipo de documento:
Article
País de afiliação:
Estados Unidos