Cheng S, Barceló J M, Korneluk R G
Department of Human Genetics, Roche Molecular Systems, Inc., Alameda, CA 94501, USA.
Hum Mutat. 1996;7(4):304-10. doi: 10.1002/(SICI)1098-1004(1996)7:4<304::AID-HUMU3>3.0.CO;2-8.
The CTG trinucleotide repeat expansions that are associated with myotonic dystrophy can be up to several thousand repeat units in length. We have developed a PCR protocol that has the potential to amplify mutant alleles with very large numbers of CTG repeats. The amplification uses the rTth DNA polymerase, XL system for long PCR targets together with primers which do not closely flank the repeat region and partial substitution of 7-deaza-dGTP for dGTP. Alleles containing up to approximately 800 CTG repeats were detected directly in agarose gels stained with ethidium bromide. Larger CTG repeat expansions required Southern blot transfer and detection with a repeat sequence probe; using this method, alleles containing up to approximately 2700 CTG repeats were detected. The PCR-based method described here was comparable to previous Southern blots of EcoRI-restriction digested genomic DNA in both the approximate size and heterogeneity of mutant alleles detected, but provided more precise sizes of the CTG repeat expansions than the restriction digest approach. This PCR protocol could potentially simplify current mutation detection protocols in the molecular diagnosis of myotonic dystrophy, and facilitate molecular studies of the disease.
与强直性肌营养不良相关的CTG三核苷酸重复序列扩增长度可达数千个重复单元。我们开发了一种聚合酶链反应（PCR）方案，该方案有潜力扩增具有大量CTG重复序列的突变等位基因。该扩增使用rTth DNA聚合酶、用于长PCR靶标的XL系统以及不紧邻重复区域侧翼的引物，并部分用7-脱氮-dGTP替代dGTP。在溴化乙锭染色的琼脂糖凝胶中可直接检测到含有多达约800个CTG重复序列的等位基因。更大的CTG重复序列扩增需要进行Southern印迹转移并用重复序列探针进行检测；使用这种方法，可检测到含有多达约2700个CTG重复序列的等位基因。此处描述的基于PCR的方法在检测到的突变等位基因的大致大小和异质性方面与先前对EcoRI酶切基因组DNA进行的Southern印迹相当，但比酶切方法能提供更精确的CTG重复序列扩增大小。这种PCR方案有可能简化目前强直性肌营养不良分子诊断中的突变检测方案，并促进对该疾病的分子研究。
