Homogeneous assays for single-nucleotide polymorphism typing using AlphaScreen
Homogeneous assays for single-nucleotide polymorphism typing using AlphaScreen
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DOI：
10.1101/gr.172501
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发表时间：
2001-04-01
期刊：
影响因子：
7
通讯作者：
Budarf, ML
中科院分区：
文献类型：
--
作者：
Beaudet, L;Bédard, J;Budarf, ML
AlphaScreen 技术允许开发高通量同质邻近分析。在这些测定中，当 680 nm 激光照射靠近受体珠的供体珠时，会产生信号。为了检测核酸，供体和受体珠子通过两个桥接探针接近，这两个桥接探针同时与共同靶标和共价连接到珠子的通用寡核苷酸杂交。该方法可以检测低至 10 摩尔的单链 DNA 靶标。 AlphaScreen 与等位基因特异性扩增 (ASA) 和等位基因特异性杂交 (ASH) 的结合使得两个同源单核苷酸多态性 (SNP) 基因分型平台得以开发。两种类型的检测都非常稳健，通常可以提供 99% 的准确基因分型结果。这两种检测方法非常简单，不需要 PCR 后操作。基因分型已在 96 孔和 384 孔格式中成功进行，体积小至 2 µl，从而大大减少了基因分型所需的试剂和基因组 DNA 的数量。这些结果表明AlphaScreen技术可以成功地适应高通量基因分型。
AlphaScreen technology allows the development of high-throughput homogeneous proximity assays. In these assays, signal is generated when 680 nm laser fight irradiates a donor bead in close proximity to an acceptor bead. For the detection of nucleic acids, donor and acceptor beads are brought into proximity by two bridging probes that hybridize simultaneously to a common target and to the generic oligonucleotides attached covalently to the beads. This method allows the detection of as little as 10 amole of a single-stranded DNA target. The combination of AlphaScreen with allele-specific amplification (ASA) and allele-specific hybridization (ASH) has allowed the development of two homogenous single-nucleotide polymorphism (SNP) genotyping platforms. Both types of assay are very robust, routinely giving accurate genotyping results with 99%. The two assays are remarkably simple, requiring no post-PCR manipulations. Genotyping has been performed successfully in 96- and 384-well formats with volumes as small as 2 mul, allowing a considerable reduction in the amount of reagents and genomic DNA necessary for genotyping. These results show that the AlphaScreen technology can be successfully adapted to high-throughput genotyping.