Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes
Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes
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DOI：
10.1038/7044
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发表时间：
1999-03-01
影响因子：
46.9
通讯作者：
Brow, MAD
中科院分区：
文献类型：
--
作者：
Lyamichev, V;Mast, AL;Brow, MAD
从古细菌中分离的瓣状核酸内切酶（FEN）显示出识别和切割当两个重叠的寡核苷酸与靶DNA链杂交时形成的结构。下游寡核苷酸探针被切割，并且切割的精确位点取决于与上游寡核苷酸的重叠量。我们已经证明，使用热稳定的古细菌FEN允许在促进探针周转的温度下进行反应，而不需要温度循环。所产生的切割信号的扩增使得能够在复杂混合物中以亚阿托摩尔水平检测特异性DNA靶标。此外，我们提供的证据表明，这种切割是足够具体的，使歧视的单碱基差异，并可以区分纯合子和杂合子在基因组DNA的单拷贝基因。
Flap endonucleases (FENs) isolated from archaea are shown to recognize and cleave a structure formed when two overlapping oligonucleotides hybridize to a target DNA strand. The downstream oligonucleotide probe is cleaved, and the precise site of cleavage is dependent on the amount of overlap with the upstream oligonucleotide. We have demonstrated that use of thermostable archaeal FENs allows the reaction to be performed at temperatures that promote probe turnover without the need for temperature cycling. The resulting amplification of the cleavage signal enables the detection of specific DNA targets at sub-attomole levels within complex mixtures. Moreover, we provide evidence that this cleavage is sufficiently specific to enable discrimination of single-base differences and can differentiate homozygotes from heterozygotes in single-copy genes in genomic DNA.