Simple and sensitive detection of mutations in the ras proto-oncogenes using PNA-mediated PCR clamping
Simple and sensitive detection of mutations in the ras proto-oncogenes using PNA-mediated PCR clamping
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DOI：
10.1093/nar/24.5.983
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发表时间：
1996-03-01
影响因子：
14.9
通讯作者：
Neubauer, A
中科院分区：
文献类型：
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作者：
Thiede, C;Bayerdorffer, E;Neubauer, A
ras原癌基因的点突变是人类恶性肿瘤中最常见的变化之一(1)，并且可能具有预后重要性。已经发表了多种检测ras突变的方法（2-4）。然而，最常用的检测方法受到灵敏度不理想（2,3）、可检测突变谱(4)或需要放射性标记（2,3）的限制。在这里，我们提出了一种检测ras点突变的新方法，该方法基于最近描述的肽核酸（PNA）介导的PCR夹紧方法(5)。PNAs是dna模拟物，其中磷酸核糖主干被（2-氨基乙基）-甘氨酸单元的肽样重复所取代。由于这种化学差异，PNA与DNA分子在几个方面存在差异：(i)与相应的DNA/DNA杂交序列相比，PNA/DNA杂交序列具有更高的热稳定性（混合序列为~ 1_C/碱基）；（ii） PNA/DNA杂交种比相应的DNA/DNA杂交种更不稳定(5)；（iii） PNAs不能作为PCR的引物分子。利用这些特征检测ras基因突变的基本思路是将最初描述的单个位置突变分析(5)扩展到在一个PCR中聚集在4-5个bp跨度内的几个突变。其原理是将染色体人DNA杂交到与野生型（wt） Ki-ras序列周围的密码子12和13互补的15mer PNA（图1中图示）。我们推断，在wt Ki-ras的情况下，PNA/DNA杂交的形成将是有利的。结合的PNA应该在空间上阻碍部分重叠的通用寡核苷酸的退火，从而使正常的Ki-ras序列无法进行充分的PCR扩增。在突变等位基因的情况下，PNA/DNA杂交体的熔化温度降低，从而使23mer寡核苷酸优于PNA退火和突变序列的优先扩增。该模型在来自几种肿瘤实体的密码子12和13的12个可能的Ki-ras突变中的6个上进行了测试，这些突变是我们可以获得的，并且已经被描述过（6,7）。发现2.84µM的PNA-1浓度足以抑制从wt DNA开始的任何可检测到的Ki-ras扩增（数据未显示）。相比之下，即使在14.2µM的PNA-1中，也没有减少
Point mutations in the ras proto-oncogenes are amongst the most frequent changes found in human malignancies (1) and may have prognostic importance. A variety of methods have been published for the detection of ras mutations (2–4). However, the most frequently used assays are limited either by an unsatisfactory sensitivity (2, 3), the spectrum of detectable mutations (4) or require radioactive labeling (2, 3). Here, we present a novel approach for the detection of ras point mutations based on the recently described method of peptide nucleic acid (PNA) mediated PCR clamping (5). PNAs are DNA-mimics where the phosphoribose backbone is replaced by a peptide-like repeat of (2-aminoethyl)-glycine units. Due to this chemical difference PNAs differ from DNA molecules in several aspects:(i) PNA/DNA-hybrids have a higher thermal stability compared with the corresponding DNA/DNA hybrids (∼ 1_C/base for mixed sequences);(ii) PNA/DNA hybrids are more destabilized by single base pair mismatches than the corresponding DNA/DNA hybrids (5); and (iii) PNAs could not serve as primer molecules in PCR.The basic idea to use these features for the detection of ras gene mutations was to extend the original assay described for mutations at a single position (5) to several mutations clustered in a 4–5 bp span in one PCR. The principle was to hybridize chromosomal human DNA to a 15mer PNA complementary to the wild-type (wt) Ki-ras sequence surrounding codons 12 and 13 (schematically illustrated in Fig. 1). We reasoned that, in the case of wt Ki-ras, formation of PNA/DNA hybrids would be favoured. The bound PNA should sterically hinder annealing of a partially overlapping generic oligonucleotide, thus excluding the normal Ki-ras sequence from sufficient PCR amplification. In the case of mutant alleles, the melting temperature of the PNA/DNA hybrid was reduced, thereby allowing the 23mer oligonucleotide to outcompete PNA annealing and preferential amplification of mutant sequences. This model was tested on six of 12 possible Ki-ras mutations in codons 12 and 13 derived from several tumor entities, which were available to us and had been characterized previously (6, 7). A concentration of 2.84 µM PNA-1 was found sufficient to inhibit any detectable Ki-ras amplification starting from wt DNA (data not shown). In contrast, even at 14.2 µM PNA-1 no reduction in