Quantitative experimental determination of primer-dimer formation risk by free-solution conjugate electrophoresis.
Quantitative experimental determination of primer-dimer formation risk by free-solution conjugate electrophoresis.
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DOI：
10.1002/elps.201100452
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发表时间：
2012-02
期刊：
影响因子：
2.9
通讯作者：
Barron, Annelise E.
中科院分区：
文献类型：
--
作者：
Desmarais, Samantha M.;Leitner, Thomas;Barron, Annelise E.
DNA条形码是短的，独特的ssDNA引物，“标记”单个生物分子。为了更好地理解限制引物-二聚体形成的生物物理参数，我们开发了一种毛细管电泳方法，该方法利用药物-标签-DNA缀合物来量化引物-条形码对之间的二聚化风险。用这种独特的自由溶液共轭电泳（FSCE）方法获得的结果是有用的定量精确的输入数据的二聚化风险的参数化计算模型。设计了一组荧光标记的模型引物-条形码缀合物，其具有不同长度的互补区域以定量异二聚化作为温度的函数。引物-二聚体情况包括两个30-mer引物，其中一个与实验室制备的、化学合成的聚-N-甲氧基乙基甘氨酸药物标签共价缀合，其降低ssDNA的电泳迁移率以将其与ds引物-二聚体区分开。拖拽标签还提供了dsDNA种类的迁移率的变化，这使得我们能够定量引物-二聚体的形成。在实验研究中，将具有完全或部分互补序列的寡核苷酸引物-条形码对退火，然后在不同温度下通过游离溶液缀合物CE分离，以评估对引物-二聚体形成的影响。当30个碱基对中少于30个被结合时，二聚化与温度呈负相关。当超过15个连续的碱基对形成时，发生二聚化，但非连续的碱基对即使在30个可能的碱基对中有20个结合时也不会产生稳定的二聚体。使用自由溶液电泳结合类肽拖曳标签和不同的荧光团，使短的DNA片段的精确分离，建立一个新的迁移率变动测定检测引物二聚体的形成。
DNA barcodes are short, unique ssDNA primers that “mark” individual biomolecules. To gain better understanding of biophysical parameters constraining primer-dimer formation between primers that incorporate barcode sequences, we have developed a capillary electrophoresis method that utilizes drag-tag-DNA conjugates to quantify dimerization risk between primer-barcode pairs. Results obtained with this unique free-solution conjugate electrophoresis (FSCE) approach are useful as quantitatively precise input data to parameterize computation models of dimerization risk. A set of fluorescently labeled, model primer-barcode conjugates were designed with complementary regions of differing lengths to quantify heterodimerization as a function of temperature. Primer-dimer cases comprised two 30-mer primers, one of which was covalently conjugated to a lab-made, chemically synthesized poly-N-methoxyethylglycine drag-tag, which reduced electrophoretic mobility of ssDNA to distinguish it from ds primer-dimers. The drag-tags also provided a shift in mobility for the dsDNA species, which allowed us to quantitate primer-dimer formation. In the experimental studies, pairs of oligonucleotide primer-barcodes with fully or partially complementary sequences were annealed, and then separated by free-solution conjugate CE at different temperatures, to assess effects on primer-dimer formation. When less than 30 out of 30 basepairs were bonded, dimerization was inversely correlated to temperature. Dimerization occurred when more than 15 consecutive basepairs formed, yet non-consecutive basepairs did not create stable dimers even when 20 out of 30 possible basepairs bonded. The use of free-solution electrophoresis in combination with a peptoid drag-tag and different fluorophores enabled precise separation of short DNA fragments to establish a new mobility shift assay for detection of primer-dimer formation.
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