Sensitive detection of polyalanine expansions in PHOX2B by polymerase chain reaction using bisulfite-converted DNA
Sensitive detection of polyalanine expansions in PHOX2B by polymerase chain reaction using bisulfite-converted DNA
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DOI：
10.1016/s1525-1578(10)60598-3
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发表时间：
2005-11-01
影响因子：
4.1
通讯作者：
Hayasaka, K
中科院分区：
文献类型：
--
作者：
Horiuchi, H;Sasaki, A;Hayasaka, K
先天性中枢性低通气综合征，也称为Ondine的诅咒，其特征是睡眠期间的特发性异常呼吸控制。最近的研究表明，PHOX 2B的聚丙氨酸扩增与这种疾病的发病机制有关。然而，它是难以检测的重复道，因为它的高GC含量抑制常规的聚合酶链反应（PCR）扩增。在这里，我们描述了一种亚硫酸氢盐处理的DNA，其中尿嘧啶是通过脱氨基的未甲基化的胞嘧啶残基。DNA脱氨允许直接PCR扩增，通过测序将C替换为T，产生了常见的20个残基重复序列的123 bp产物。它解决了伴随着扩增等位基因扩增不足的等位基因缺失。在以前的研究中，10例先天性中枢性通气不足综合征患者中有9例的扩张检测率显著提高。PCR扩增前DNA的化学转化有利于有效检测富含GC的聚丙氨酸片段。
Congenital central hypoventilation syndrome, also known as Ondine's curse, is characterized by idiopathic abnormal control of respiration during sleep. Recent studies indicate that a polyalanine expansion of PHOX2B is relevant to the pathogenesis of this disorder. However, it is difficult to detect the repeated tract because its high GC content inhibits conventional polymerase chain reaction (PCR) amplification. Here, we describe a bisulfite treatment for DNA in which uracil is obtained by deamination of unmethylated cytosine residues. Deamination of DNA permitted direct PCR amplification that yielded a product of 123 bp for the common 20-residue repetitive tract with replacement of C with T by sequencing. it settled allele dropouts accompanied by insufficient amplification of expanded alleles. The defined procedure dramatically improved detection of expansions to 9 of 10 congenital central hypoventilation syndrome patients examined in a previous study. The chemical conversion of DNA before PCR amplification facilitates effective detection of GC-rich polyalanine tracts.