Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR.
Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR.
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DOI：
10.1007/s00216-018-0931-z
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发表时间：
2018-04
影响因子：
4.3
通讯作者：
Rådström P
中科院分区：
文献类型：
--
作者：
Sidstedt M;Hedman J;Romsos EL;Waitara L;Wadsö L;Steffen CR;Vallone PM;Rådström P
血液样品广泛用于基于PCR的DNA分析，例如传染病诊断、癌症诊断和法医遗传学领域。在这项研究中，通过在数字PCR和实时PCR中使用全血以及已知的PCR抑制分子来评估血液诱导的PCR抑制背后的机制。此外，电泳迁移率变动分析被应用于研究抑制蛋白和DNA之间的相互作用，和等温滴定量热法被用来直接测量对DNA聚合酶活性的影响。全血导致阳性数字PCR反应数量减少，扩增效率降低，并导致被动参比染料6-羧基-X-罗丹明以及双链DNA结合染料EvaGreen的荧光严重淬灭。发现免疫球蛋白G与单链基因组DNA结合，导致定量循环值增加。血红蛋白影响DNA聚合酶活性，从而降低扩增效率。血红蛋白和血红素被证明是血液中负责荧光猝灭的分子。总之，血红蛋白和免疫球蛋白G是血液中两种主要的PCR抑制剂，其中第一种通过对DNA聚合酶活性的直接作用来影响扩增，并淬灭游离染料分子的荧光，而后者与单链基因组DNA结合，在最初的几个PCR循环中阻碍DNA聚合。血红蛋白和免疫球蛋白G（IgG）的PCR抑制机制。Cq定量循环，dsDNA双链DNA，ssDNA单链DNA本文的在线版本（10. 1007/s 00216 -018-0931-z）包含补充材料，可供授权用户使用。
Blood samples are widely used for PCR-based DNA analysis in fields such as diagnosis of infectious diseases, cancer diagnostics, and forensic genetics. In this study, the mechanisms behind blood-induced PCR inhibition were evaluated by use of whole blood as well as known PCR-inhibitory molecules in both digital PCR and real-time PCR. Also, electrophoretic mobility shift assay was applied to investigate interactions between inhibitory proteins and DNA, and isothermal titration calorimetry was used to directly measure effects on DNA polymerase activity. Whole blood caused a decrease in the number of positive digital PCR reactions, lowered amplification efficiency, and caused severe quenching of the fluorescence of the passive reference dye 6-carboxy-X-rhodamine as well as the double-stranded DNA binding dye EvaGreen. Immunoglobulin G was found to bind to single-stranded genomic DNA, leading to increased quantification cycle values. Hemoglobin affected the DNA polymerase activity and thus lowered the amplification efficiency. Hemoglobin and hematin were shown to be the molecules in blood responsible for the fluorescence quenching. In conclusion, hemoglobin and immunoglobulin G are the two major PCR inhibitors in blood, where the first affects amplification through a direct effect on the DNA polymerase activity and quenches the fluorescence of free dye molecules, and the latter binds to single-stranded genomic DNA, hindering DNA polymerization in the first few PCR cycles. PCR inhibition mechanisms of hemoglobin and immunoglobulin G (IgG). Cq quantification cycle, dsDNA double-stranded DNA, ssDNA single-stranded DNA The online version of this article (10.1007/s00216-018-0931-z) contains supplementary material, which is available to authorized users.
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影响因子：
3.1
作者：
Green, Robert L.;Lagace, Robert E.;Mulero, Julio J.
通讯作者：
Mulero, Julio J.
影响因子：
2.7
作者：
Hedman, Johannes;Nordgaard, Anders;Radstrom, Peter
通讯作者：
Radstrom, Peter
影响因子：
3.1
作者：
Gray, Kerryn;Crowle, Damian;Scott, Pam
通讯作者：
Scott, Pam
影响因子：
5.2
作者：
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通讯作者：
Hogrefe H
影响因子：
9.4
作者：
Abu al-Soud, W;Rådström, P
通讯作者：
Rådström, P