Levedakou E N, Landegren U, Hood L E
Division of Biology, California Institute of Technology, Pasadena 91125.
Biotechniques. 1989 May;7(5):438-42.
A two-step strategy is described here to rapidly analyze gene-sequence variation or polymorphism. First, DNA sequences flanking the coding region of the gene to be analyzed are determined directly from a cosmid clone, including the gene, using the modified T7 DNA polymerase and sequencing primers based on the cDNA sequence of the gene. Second, the identified gene-flanking sequences are used to design amplification primers for the polymerase chain reaction (PCR) to permit amplification of DNA segments of up to 1 kilobase in genomic DNA from multiple individuals. These amplified DNA segments are directly sequenced using the thermostable Taq DNA polymerase.
本文描述了一种用于快速分析基因序列变异或多态性的两步策略。首先，使用改良的T7 DNA聚合酶和基于该基因cDNA序列的测序引物，直接从包含该基因的黏粒克隆中确定待分析基因编码区两侧的DNA序列。其次，将鉴定出的基因侧翼序列用于设计聚合酶链反应（PCR）的扩增引物，以扩增来自多个个体的基因组DNA中长达1千碱基的DNA片段。这些扩增的DNA片段使用热稳定的Taq DNA聚合酶直接测序。
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