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在实时荧光定量PCR中可以观察到什么,以及何时会显现出来?

What can be observed in real time PCR and when does it show?

作者信息

Chigansky Pavel, Jagers Peter, Klebaner Fima C

机构信息

Department of Statistics, The Hebrew University, Mount Scopus, 91905, Jerusalem, Israel.

Mathematical Sciences, Chalmers University of Technology and University of Gothenburg, 412 96, Gothenburg, Sweden.

出版信息

J Math Biol. 2018 Feb;76(3):679-695. doi: 10.1007/s00285-017-1154-1. Epub 2017 Jun 30.

DOI:10.1007/s00285-017-1154-1
PMID:28667372
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5772144/
Abstract

Real time, or quantitative, PCR typically starts from a very low concentration of initial DNA strands. During iterations the numbers increase, first essentially by doubling, later predominantly in a linear way. Observation of the number of DNA molecules in the experiment becomes possible only when it is substantially larger than initial numbers, and then possibly affected by the randomness in individual replication. Can the initial copy number still be determined? This is a classical problem and, indeed, a concrete special case of the general problem of determining the number of ancestors, mutants or invaders, of a population observed only later. We approach it through a generalised version of the branching process model introduced in Jagers and Klebaner (J Theor Biol 224(3):299-304, 2003. doi: 10.1016/S0022-5193(03)00166-8 ), and based on Michaelis-Menten type enzyme kinetical considerations from Schnell and Mendoza (J Theor Biol 184(4):433-440, 1997). A crucial role is played by the Michaelis-Menten constant being large, as compared to initial copy numbers. In a strange way, determination of the initial number turns out to be completely possible if the initial rate v is one, i.e all DNA strands replicate, but only partly so when [Formula: see text], and thus the initial rate or probability of succesful replication is lower than one. Then, the starting molecule number becomes hidden behind a "veil of uncertainty". This is a special case, of a hitherto unobserved general phenomenon in population growth processes, which will be adressed elsewhere.

摘要

实时定量PCR通常从非常低浓度的初始DNA链开始。在迭代过程中,数量会增加,首先基本上是翻倍,之后主要呈线性增加。只有当实验中的DNA分子数量远大于初始数量时,才有可能观察到其数量,此时可能会受到个体复制随机性的影响。初始拷贝数还能确定吗?这是一个经典问题,实际上是一个确定仅在后期观察到的群体的祖先、突变体或入侵者数量这一一般问题的具体特殊情况。我们通过Jagers和Klebaner(《理论生物学杂志》224(3):299 - 304,2003年。doi: 10.1016/S0022 - 5193(03)00166 - 8)中引入的分支过程模型的广义版本来处理这个问题,并基于Schnell和Mendoza(《理论生物学杂志》184(4):433 - 440,1997年)的米氏酶动力学考虑。与初始拷贝数相比,米氏常数较大起着关键作用。以一种奇怪的方式,如果初始速率v为1,即所有DNA链都复制,那么确定初始数量是完全可能的,但当[公式:见原文]时,情况就只是部分如此,因此成功复制的初始速率或概率低于1。那么,起始分子数就会隐藏在“不确定性的面纱”之后。这是群体增长过程中一种迄今未被观察到的普遍现象的特殊情况,将在其他地方进行探讨。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a45d/5772144/8ca0335ae7dd/285_2017_1154_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a45d/5772144/8ca0335ae7dd/285_2017_1154_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a45d/5772144/8ca0335ae7dd/285_2017_1154_Fig1_HTML.jpg

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本文引用的文献

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Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing.基于聚合酶链式反应(PCR)的简单、多重DNA条形码技术能够通过测序在液体活检中实现灵敏的突变检测。
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一种针对大群体中选择性清除分布的简单半确定性近似方法。
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Nucleic Acids Res. 2012 Jan;40(2):e10. doi: 10.1093/nar/gkr775. Epub 2011 Nov 18.
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Nucleic Acids Res. 2004 Mar 29;32(6):e56. doi: 10.1093/nar/gnh053.
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