Molecular crowding increases the amplification success of multiple displacement amplification and short tandem repeat genotyping
Molecular crowding increases the amplification success of multiple displacement amplification and short tandem repeat genotyping
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DOI：
10.1016/j.ab.2006.04.039
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发表时间：
2006-08-15
影响因子：
2.9
通讯作者：
Koukoulas, Irene
中科院分区：
文献类型：
--
作者：
Ballantyne, Kaye N.;van Oorschot, Roland A. H.;Koukoulas, Irene
多重置换扩增（MDA）可以从少量的模板产生大量的基因组DNA产物。我们研究了MDA扩增含有非常少量的靶DNA（5 pg至1 ng）的样品的能力，在存在第二个较大的DNA样品的下游短串联重复序列（STR）多重基因分型。我们观察到，当与仅标准PCR或仅含有单一痕量DNA样品的MDA相比时，在这些混合样品中次要部分的STR扩增成功率增加。检测到等位基因数量增加，基因座之间的扩增偏倚小于经历相同方案的单一来源样本。为了提高STR基因分型的准确性，用动物DNA代替额外的人DNA，保持扩增的人特异性STR基因座的数量和质量的增加。聚乙二醇400是一种常用的拥挤剂，在MDA反应中用作添加的基因组DNA的替代物，并产生非常相似的结果。因此，我们认为，额外的DNA是作为一个分子拥挤剂在MDA。在拥挤的条件下对痕量DNA进行MDA导致更大数量的等位基因被扩增，并且等位基因之间发生更平衡的扩增。(c)2006年爱思唯尔公司All rights reserved.
Multiple displacement amplification (MDA) can generate large quantities of genomic DNA product from small amounts of template. We investigated the ability of MDA to amplify samples containing very small amounts of target DNA (5 pg to 1 ng) in the presence of a second, larger DNA sample for downstream short tandem repeat (STR) multiplex genotyping. We observed that STR amplification success of the minor fraction was increased in these mixed samples when compared with standard PCR only or MDA containing only the single trace DNA sample. Increased numbers of alleles were detected, with less amplification bias between loci than in single source samples undergoing the same protocol. To improve the STR genotyping accuracy, animal DNA was substituted for the additional human DNA, maintaining the increase in the number and quality of human-specific STR loci amplified. Polyethylene glycol 400, a commonly used crowding agent, was used as a replacement for the added genomic DNA in the MDA reaction and produced very similar results. Therefore, we suggest that additional DNA is acting as a molecular crowding agent during MDA. Performing MDA on trace amounts of DNA under crowded conditions results in greater numbers of alleles being amplified and more balanced amplification occurring between alleles. (c) 2006 Elsevier Inc. All rights reserved.