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首页> 外文期刊>Nucleic Acids Research >Multiplex SNP typing by bioluminometric assay coupled with terminator incorporation (BATI)
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Multiplex SNP typing by bioluminometric assay coupled with terminator incorporation (BATI)

机译：通过生物发光测定结合终止子掺入（BATI）进行多重SNP分型

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摘要

A multiplex single-nucleotide polymorphism (SNP) typing platform using 'bioluminometric assay coupled with terminator [2',3'-dideoxynucleoside triphosphates (ddNTPs)] incorporation' (named 'BATI' for short) was developed. All of the reactions are carried out in a single reaction chamber containing target DNAs, DNA polymerase, reagents necessary for converting PPi into ATP and reagents for luciferase reaction. Each of the four ddNTPs is dispensed into the reaction chamber in turn. PPi is released by a nucleotide incorporation reaction and is used to produce ATP when the ddNTP dispensed is complementary to the base in a template. The ATP is used in a luciferase reaction to release visible light. Only 1 nt is incorporated into a template at a time because ddNTPs do not have a 3' hydroxyl group. This feature greatly simplifies a sequencing spectrum. The luminescence is proportional to the amount of template incorporated. Only one peak appears in the spectrum of a homozygote sample, and two peaks at the same intensity appear for a heterozygote sample. In comparison with pyrosequencing using dNTP, the spectrum obtained by BATI is very simple, and it is very easy to determine SNPs accurately from it. As only one base is extended at a time and the extension signals are quantitative, the observed spectrum pattern is uniquely determined even for a sample containing multiplex SNPs. We have successfully used BATI to type various samples containing plural target sequence areas. The measurements can be carried out with an inexpensive and small luminometer using a photodiode array as the detector. It takes only a few minutes to determine multiplex SNPs. These results indicate that this novel multiplexed approach can significantly decrease the cost of SNP typing and increase the typing throughput with an inexpensive and small luminometer.
机译：开发了一个多重单核苷酸多态性（SNP）分型平台，该平台使用了“生物发光测定结合终止子[2'，3'-二氧脱氧核苷三磷酸（ddNTPs）]掺入”（简称为“ BATI”）。所有反应都在一个包含目标DNA，DNA聚合酶，将PPi转换为ATP所需的试剂以及萤光素酶反应试剂的单一反应室内进行。四个ddNTP中的每一个依次分配到反应室中。 PPi通过核苷酸掺入反应释放，并在分配的ddNTP与模板中的碱基互补时用于产生ATP。 ATP用于萤光素酶反应中以释放可见光。一次仅将1 nt掺入模板中，因为ddNTP不具有3'羟基。此功能大大简化了测序范围。发光与掺入的模板的量成比例。纯合子样品的光谱中仅出现一个峰，杂合子样品的强度相同时出现两个峰。与使用dNTP进行焦磷酸测序相比，BATI获得的光谱非常简单，并且很容易从中准确确定SNP。由于一次仅扩展一个碱基，并且扩展信号是定量的，因此即使对于包含多重SNP的样品，也可以唯一确定观察到的光谱模式。我们已经成功地使用BATI来键入包含多个目标序列区域的各种样本。可以使用光电二极管阵列作为检测器，通过廉价，小型的发光计进行测量。确定多重SNP只需几分钟。这些结果表明，这种新颖的多路复用方法可以显着降低SNP打字的成本，并通过廉价且小巧的发光计提高打字吞吐量。

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