PNA Clamp Technique for Detecting a Ki-ras2 Mutation Using the LightCycler Instrument
PNA Clamp Technique for Detecting a Ki-ras2 Mutation Using the LightCycler Instrument
复制标题
使用 LightCycler 仪器检测 Ki-ras2 突变的 PNA 钳技术
DOI：
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发表时间：
2002
期刊：
影响因子：
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通讯作者：
G. Lahr
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文献类型：
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作者：
G. Lahr
根据文献，Ki-ras 2基因突变与约50%的结肠腺癌有关[1]。该基因编码21 kDa GTP结合蛋白，其控制细胞生长和分化的机制[2]。Ki-ras 2基因通过密码子12、13或61中的点突变转化为活性癌基因，该点突变位于可能参与GTP结合的区域。由于样品材料通常含有不同量的未受影响的野生型细胞，因此这些点突变的检测在许多研究应用中是一个问题。在微小残留病（MRD）中，只有少数细胞必须被检测到。例如，通过人工限制性片段长度多态性（aRFLP）分析密码子12 Ki-ras 2点突变非常耗时[3-5]。因此，将PNA寡聚体与LightCycler杂交探针结合应用，是检测研究样本中点突变的快速便捷方法。PNA与互补序列的结合比DNA或RNA更紧密，并且不能被Taq DNA聚合酶延伸。最初，Thiede et.等人描述了使用野生型特异性PNA寡聚体防止野生型Ki-ras 2染色体DNA的PCR扩增的方法[6]。最近，对该方法进行了修改，以与LightCycler仪器和杂交探针结合使用[7]。现在，PNA与扩增的Ki-ras 2序列结合，并通过与突变特异性杂交探针引物（传感器）竞争野生型序列来降低互补野生型序列的扩增（图1）。在这种配置中，仅获得突变体特异性信号，允许鉴定样品中存在的突变。在此，PNA夹持与杂交探针组合的使用在格鲁吉亚Lahr第一医学部，München Harlaching市立教学医院，München，德国通讯作者：G. extern.lrz-muenchen.de使用LightCycler仪器检测Ki-ras 2突变的PNA钳技术
According to the literature, mutations in the Ki-ras2 gene have been implicated in approximately 50 % of colon adenocarcinomas [1]. The gene codes for a 21 kDa GTP-binding protein which controls the mechanisms of cell growth and differentiation [2]. The Ki-ras2 gene is converted to an active oncogene by point mutations in codons 12, 13, or 61, in a region that may be involved in GTP binding. Since sample material normally contains different amounts of unaffected wild-type cells, the detection of these point mutations represents a problem in many research applications. In Minimal Residual Disease (MRD), only a few cells exist that must be detected. For example, analyzing the codon 12 Ki-ras2 point mutation by artificial restriction fragment length polymorphism (aRFLP) is very time-consuming [3–5]. Therefore, the application of a PNA oligomer, in combination with LightCycler Hybridization Probes, is a quick and convenient way to detect point mutations in research samples. PNA binds the complementary sequences tighter than DNA or RNA, and cannot be extended by Taq DNA polymerase. Originally, Thiede et. al. described a method to prevent PCR amplification of wild-type Ki-ras2 chromosomal DNA using a wild typespecific PNA oligomer [6]. Recently, this method was modified for use with the LightCycler Instrument in combination with Hybridization Probes [7]. Now, PNA binds to the amplified Ki-ras2 sequence and lowers the amplification of the complementary wild-type sequence by competing with the mutation-specific hybridization probe primer (sensor) for the wild-type sequence (Figure 1). In this configuration, only the mutant-specific signal is obtained, allowing the identification of the mutation present in the sample. Here, the use of PNA clamping in combination with Hybridization Probes in Georgia Lahr 1st Medical Dep., Municipal and Teaching Hospital München Harlaching, München, Germany Corresponding author: G.Lahr@extern.lrz-muenchen.de PNA Clamp Technique for Detecting a Ki-ras2 Mutation Using the LightCycler Instrument