Benjamin D, Van Bakel I, Craig I W
Genetics Unit, University of Oxford, UK.
Eur J Hum Genet. 2000 Feb;8(2):103-8. doi: 10.1038/sj.ejhg.5200427.
We present a novel RT-PCR-based approach for determining the inactivation status of X-linked genes. Using cDNA from cloned female cell lines in which only the maternal or paternally derived X chromosome is active, we are able to demonstrate expression from only one allele in genes known to be inactivated. Following reverse transcription, amplification across a polymorphism will yield a product from a single allele if the gene of interest is inactivated, and products from both alleles in a gene escaping inactivation. We have verified this approach using the human androgen receptor and FMR1 loci which have been shown to be subjected to normal inactivation. The potential for widespread application of this approach was shown by the successful demonstration of inactivation at the MAOA and HPRT loci using intronic polymorphisms.
我们提出了一种基于逆转录聚合酶链反应（RT-PCR）的新方法，用于确定X连锁基因的失活状态。利用来自克隆雌性细胞系的互补DNA（cDNA），其中只有母源或父源的X染色体是活跃的，我们能够证明已知失活基因中只有一个等位基因表达。逆转录后，如果感兴趣的基因失活，跨越多态性的扩增将产生来自单个等位基因的产物；而在逃避失活的基因中，则会产生来自两个等位基因的产物。我们已经使用人雄激素受体和脆性X智力低下基因1（FMR1）位点验证了这种方法，这些位点已被证明会发生正常失活。通过利用内含子多态性成功证明单胺氧化酶A（MAOA）和次黄嘌呤-鸟嘌呤磷酸核糖转移酶（HPRT）位点的失活，表明了这种方法广泛应用的潜力。
