Droplet digital PCR technology promises new applications and research areas
Droplet digital PCR technology promises new applications and research areas
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DOI：
10.3109/19401736.2014.913168
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发表时间：
2016-01
影响因子：
--
通讯作者：
P. Manoj;R. Gandhi
中科院分区：
文献类型：
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作者：
P. Manoj;R. Gandhi
摘要 数字聚合酶链式反应（dPCR）用于核酸定量，其应用包括低水平病原体、稀有基因序列的检测和精确定量、拷贝数变异、稀有突变的定量以及相关基因表达。这里，PCR在大量反应室或分区中进行，并且反应在每个分区中单独进行。这种分离可以更可靠地收集和灵敏地测量核酸。通过计算扩增的目标序列（阳性液滴）和未扩增的分区数（阴性液滴）来计算结果。通过泊松算法计算目标序列的平均数。泊松校正可补偿任何液滴中存在的多个目标基因拷贝。该方法提供的信息准确、精确，与 qPCR 相比，重现性高，且不易受抑制剂影响。它已在研究基因序列变异（例如拷贝数变异和点突变）、区分几乎相同等位基因的表达之间的差异、评估临床相关遗传变异方面得到证明，并且通常用于 NGS 方法样品的克隆扩增。 dPCR 通过使用参考标准化进行绝对定量，可以更可靠地预测肿瘤状态和患者预后。液滴数字 PCR 可以准确检测与一系列疾病和病症以及衰老相关的罕见线粒体 DNA 缺失。
Abstract Digital Polymerase Chain Reaction (dPCR) is used to quantify nucleic acids and its applications are in the detection and precise quantification of low-level pathogens, rare genetic sequences, quantification of copy number variants, rare mutations and in relative gene expressions. Here the PCR is performed in large number of reaction chambers or partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid. Results are calculated by counting amplified target sequence (positive droplets) and the number of partitions in which there is no amplification (negative droplets). The mean number of target sequences was calculated by Poisson Algorithm. Poisson correction compensates the presence of more than one copy of target gene in any droplets. The method provides information with accuracy and precision which is highly reproducible and less susceptible to inhibitors than qPCR. It has been demonstrated in studying variations in gene sequences, such as copy number variants and point mutations, distinguishing differences between expression of nearly identical alleles, assessment of clinically relevant genetic variations and it is routinely used for clonal amplification of samples for NGS methods. dPCR enables more reliable predictors of tumor status and patient prognosis by absolute quantitation using reference normalizations. Rare mitochondrial DNA deletions associated with a range of diseases and disorders as well as aging can be accurately detected with droplet digital PCR.