ATTACHMENT OF A 40-BASE-PAIR G+C-RICH SEQUENCE (GC-CLAMP) TO GENOMIC DNA FRAGMENTS BY THE POLYMERASE CHAIN-REACTION RESULTS IN IMPROVED DETECTION OF SINGLE-BASE CHANGES
ATTACHMENT OF A 40-BASE-PAIR G+C-RICH SEQUENCE (GC-CLAMP) TO GENOMIC DNA FRAGMENTS BY THE POLYMERASE CHAIN-REACTION RESULTS IN IMPROVED DETECTION OF SINGLE-BASE CHANGES
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DOI：
10.1073/pnas.86.1.232
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发表时间：
1989-01-01
影响因子：
11.1
通讯作者：
MYERS, RM
中科院分区：
文献类型：
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作者：
SHEFFIELD, VC;COX, DR;MYERS, RM
变性梯度凝胶电泳（DGGE）可用于区分两个DNA分子，其差异小至单个碱基取代。此方法检测. apprxeq。DNA片段中所有可能的单碱基变化的50%，范围从50至一千个碱基对。为了增加可通过DGGE区分的单碱基变化的数量，我们使用聚合酶链式反应将40个碱基对的富含G + C的序列（称为GC-钳）连接到扩增的DNA片段的一端，所述扩增的DNA片段包含小鼠和人β-葡萄糖受体的区域。珠蛋白基因我们表明，该GC-钳允许检测突变，包括人β-葡萄糖受体内的血红蛋白镰状（HbS）和血红蛋白C（HbC）突变。珠蛋白基因，这是以前无法区分的DGGE。除了提供一种将GC钳连接到基因组DNA片段的简单方法外，聚合酶链反应技术还大大提高了DGGE的灵敏度。通过这种方法，可以通过溴化乙锭染色凝胶来检测来源于< 5 ng人类基因组DNA的DNA片段，从而避免了对放射性探针的需要。这些改进扩展了DGGE用于检测基因组和克隆DNA中的多态性和突变的适用性。
Denaturing gradient gel electrophoresis (DGGE) can be used to distinguish two DNA molecules that differ by as little as a single-base substitution. This method detects .apprxeq. 50% of all possible single-base changes in DNA fragments ranging from 50 to .apprxeq. 1000 base pairs. To increase the number of single-base changes that can be distinguished by DGGE, we used the polymerase chain reaction to attach a 40-base-pair G + C-rich sequence, designated a GC-clamp, to one end of amplified DNA fragments that encompass regions of the mouse and human .beta.-globin genes. We show that this GC-clamp allows the detection of mutations, including the hemoglobin sickle (HbS) and hemoglobin C (HbC) mutations within the human .beta.-globin gene, that were previously indistinguishable by DGGE. In addition to providing an easy way to attach a GC-clamp to genomic DNA fragments, the polymerase chain reaction technique greatly increases the sensitivity of DGGE. With this approach, DNA fragments derived from < 5 ng of human genomic DNA can be detected by ethidium bromide staining of the gel, obviating the need for radioactive probes. These improvements extend the applicability of DGGE for the detection of polymorphisms and mutations in genomic and cloned DNA.