Hsiao K M, Lin H M, Pan H, Li T C, Chen S S, Jou S B, Chiu Y L, Wu M F, Lin C C, Li S Y
Department of Life Sciences, Chung Shan Medical and Dental College, Taichung, Taiwan.
J Clin Lab Anal. 1999;13(4):188-93. doi: 10.1002/(sici)1098-2825(1999)13:4<188::aid-jcla8>3.0.co;2-g.
Myotonic dystrophy (DM) is caused by a CTG trinucleotide expansion mutation at exon 15 of the myotonic dystrophy protein kinase gene. The clinical severity of this disease correlates with the length of the CTG trinucleotide repeats. Determination of the CTG repeat length has been primarily relied on by Southern blot analysis of restriction enzyme-digested genomic DNA. The development of PCR-based Southern blotting methodology provides a much more sensitive and simpler protocol for DM diagnosis. However, the quality of the template and the high (G+C) ratio of the amplified region hamper the use of PCR on the diagnosis of DM. A modified PCR protocol to amplify different lengths of CTG repeat region using various concentrations of 7deaza-dGTP has been reported (1). Here we describe a procedure including sample collection, DNA purification, and PCR analysis of CTG repeat length without using 7-deaza-dGTP. This protocol is very sensitive and convenient because only a small number of nucleate cells are needed for detection of CTG expansion. Therefore, it could be very useful in clinical and prenatal diagnosis as well as in prevalence study of DM.
强直性肌营养不良（DM）由强直性肌营养不良蛋白激酶基因第15外显子的CTG三核苷酸扩增突变引起。该疾病的临床严重程度与CTG三核苷酸重复序列的长度相关。CTG重复序列长度的测定主要依赖于对限制性内切酶消化的基因组DNA进行Southern印迹分析。基于PCR的Southern印迹方法的发展为DM诊断提供了一种更灵敏、更简便的方案。然而，模板质量和扩增区域的高（G+C）比例阻碍了PCR在DM诊断中的应用。已有报道一种改良的PCR方案，使用不同浓度的7-脱氮-dGTP扩增不同长度的CTG重复区域（1）。在此，我们描述了一种无需使用7-脱氮-dGTP的程序，包括样本采集、DNA纯化以及CTG重复序列长度的PCR分析。该方案非常灵敏且方便，因为检测CTG扩增仅需少量有核细胞。因此，它在DM的临床诊断、产前诊断以及患病率研究中可能非常有用。
