Department of Bioengineering, Stanford University, Stanford, CA 94305-5444, USA.
Electrophoresis. 2012 Feb;33(3):483-91. doi: 10.1002/elps.201100452. Epub 2012 Jan 10.
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 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 base-pairs were bonded, dimerization was inversely correlated to temperature. Dimerization occurred when more than 15 consecutive base-pairs formed, yet non-consecutive base-pairs did not create stable dimers even when 20 out of 30 possible base-pairs 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.
DNA 条码是短的、独特的 ssDNA 引物，“标记”个体生物分子。为了更好地了解限制包含条码序列的引物之间形成引物二聚体的生物物理参数，我们开发了一种毛细管电泳方法，该方法利用拖曳标签-DNA 缀合物来量化引物条码对之间的二聚化风险。利用这种独特的游离溶液缀合电泳方法获得的结果可作为定量精确的输入数据，用于参数化二聚化风险计算模型。设计了一组荧光标记的模型引物条码缀合物，其互补区域的长度不同，以量化异二聚体作为温度的函数。引物二聚体的情况包括两个 30 -mer 引物，其中一个与实验室制造的、化学合成的聚-N-甲氧基乙基甘氨酸拖曳标签共价连接，该标签降低了 ssDNA 的电泳迁移率，以将其与 ds 引物二聚体区分开来。拖曳标签还为 dsDNA 物种提供了迁移率的变化，这使我们能够定量形成引物二聚体。在实验研究中，退火具有完全或部分互补序列的寡核苷酸引物条码对，然后在不同温度下通过游离溶液缀合 CE 分离，以评估对引物二聚体形成的影响。当少于 30 个碱基对键合时，二聚化与温度呈反比。当形成超过 15 个连续碱基对时发生二聚化，但非连续碱基对即使形成 30 个可能碱基对中的 20 个也不会形成稳定的二聚体。游离溶液电泳与肽聚糖拖曳标签和不同荧光团的结合使用能够精确分离短 DNA 片段，建立用于检测引物二聚体形成的新迁移率变化分析。
