Feener C A, Boyce F M, Kunkel L M
Division of Genetics, Howard Hughes Medical Institute, Children's Hospital, Boston, MA 02115.
Am J Hum Genet. 1991 Mar;48(3):621-7.
To identify CA repeats in genomic sequences which had been previously subcloned into plasmids, we performed PCR using a (CA)n primer and a flanking vector primer on the genomic inserts. By incorporation of a restriction enzyme site into the (CA)n primer, we have been able to subclone the genomic DNA so that the sequence flanking the CA repeat is readily determined. Primers can then be designed to amplify across the CA repeat in patient DNA samples. Application of this technique to genomic DNAs surrounding the upstream "brain" promoter of the dystrophin gene has led to the discovery of four new CA repeats. Three of these repeats are highly polymorphic, with PICs ranging from .586 to .768. The location of these markers at the extreme 5' terminus of the dystrophin gene, together with their high degree of polymorphism and ease of assay, makes them ideal for linkage analysis in families with Duchenne muscular dystrophy.
为了鉴定先前已亚克隆到质粒中的基因组序列中的CA重复序列，我们使用（CA）n引物和侧翼载体引物对基因组插入片段进行PCR。通过将一个限制酶位点掺入（CA）n引物中，我们得以亚克隆基因组DNA，从而能够轻松确定CA重复序列侧翼的序列。然后可以设计引物来扩增患者DNA样本中的CA重复序列。将该技术应用于肌营养不良蛋白基因上游“脑”启动子周围的基因组DNA，已发现四个新的CA重复序列。其中三个重复序列具有高度多态性，多态信息含量（PIC）范围为0.586至0.768。这些标记位于肌营养不良蛋白基因的极端5'末端，加上它们的高度多态性和易于检测，使其成为杜氏肌营养不良症家族连锁分析的理想选择。
