Fabry disease: molecular studies in Italian patients and X inactivation analysis in manifesting carriers
Fabry disease: molecular studies in Italian patients and X inactivation analysis in manifesting carriers
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DOI：
10.1136/jmg.40.8.e103
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发表时间：
2003-08-01
影响因子：
4
通讯作者：
Zammarchi, E
中科院分区：
文献类型：
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作者：
Morrone, A;Cavicchi, C;Zammarchi, E
材料与方法α-GLA酶促测定方法与文献报道一致。从淋巴细胞中分离患者及其亲属的基因组DNA和总RNA。利用Display Systems Biotech （Copenhagen, Denmark）的displaythermal - rttm从10µg总RNA合成GLA cDNA，在野生型GLA cDNA的3 ' UTR区定位寡核苷酸dT和/或特定的反义寡核苷酸引物RNA7Rest。GLA外显子、内含子-外显子边界和GLA cDNA采用以下引物进行PCR扩增：F1 forward 5 ' gcccctgaggttaatcttaa 3 ' (-67 / - 48nt)；R1反向5 ' aactgttcccgttgagactc 3 ' (IVS1+ 87/+ 68nt)；F2 forward 5 ' ggaggtacctaagtgttcat 3 ' (IVS1−58/−39nt)；R2反向5 ' agcttctgtacagaagtgc3 ' (IVS2
MATERIALS AND METHODS The α-GLA enzymatic assay was carried out as previously reported. 16 Genomic DNA and total RNA from the patients and their relatives were isolated from lymphocytes. GLA cDNA synthesis was generated from 10 µg total RNA using displayTHERMO-RTTM by Display Systems Biotech (Copenhagen, Denmark) with oligo dT and/or a specific antisense oligonucleotide primer RNA7Rest mapping in the 3′ UTR region of the wild type GLA cDNA. The GLA exons, intron-exon boundaries, and the GLA cDNA were amplified by PCR using the following primers synthesised by Roche Molecular Biochemicals (Basel, Switzerland): F1 forward 5′ gcccctgaggttaatcttaa 3′(–67/− 48nt); R1 reverse 5′ aactgttcccgttgagactc 3′(IVS1+ 87/+ 68nt); F2 forward 5′ ggaggtacctaagtgttcat 3′(IVS1− 58/− 39nt); R2 reverse 5′ agcttctgtacagaagtgct 3′(IVS2