Xiao Pengfeng, Huang Huan, Zhou Guohua, Lu Zuhong
State Key Laboratory of Bioelectronics, Southeast University, Nanjing, PR China.
Electrophoresis. 2007 Jun;28(12):1903-12. doi: 10.1002/elps.200600794.
A novel two-step process was developed to prepare ssDNA templates for pyrosequencing. First, PCR-amplified DNA templates modified with an acrylamide group and acrylamide monomers were copolymerized in 0.1 M NaOH solution to form polyacrylamide gel spots. Second, ssDNA templates for pyrosequencing were prepared by removing electrophoretically unbound complementary strands, unmodified PCR primers, inorganic pyrophosphate (PPi), and excess deoxyribonucleotides under alkali conditions. The results show that the 3-D polyacrylamide gel network has a high immobilization capacity and the modified PCR fragments are efficiently captured. After electrophoresis, gel spots copolymerized from 10 microL of the crude PCR products and the acrylamide monomers contain template molecules on the order of pmol, which generate enough light to be detected by a regular photomultiplier tube. The porous structure of gel spots facilitated the fast transportation of the enzyme, dNTPs and other reagents, and the solution-mimicking microenvironment guaranteed polymerase efficiency for pyrosequencing. Successful genotyping from the crude PCR products was demonstrated. This method can be applied in any laboratory; it is cheap, fast, simple, and has the potential to be incorporated into a DNA-chip format for high-throughput pyrosequencing analysis.
开发了一种新颖的两步法来制备用于焦磷酸测序的单链DNA模板。首先，将用丙烯酰胺基团修饰的PCR扩增DNA模板与丙烯酰胺单体在0.1 M NaOH溶液中共聚，形成聚丙烯酰胺凝胶斑点。其次，通过在碱性条件下电泳去除未结合的互补链、未修饰的PCR引物、无机焦磷酸（PPi）和过量的脱氧核糖核苷酸，制备用于焦磷酸测序的单链DNA模板。结果表明，三维聚丙烯酰胺凝胶网络具有高固定能力，并且修饰的PCR片段被有效捕获。电泳后，由10微升粗PCR产物和丙烯酰胺单体共聚而成的凝胶斑点含有皮摩尔级别的模板分子，产生的光足以被普通光电倍增管检测到。凝胶斑点的多孔结构促进了酶、dNTP和其他试剂的快速运输，并且模拟溶液的微环境保证了焦磷酸测序的聚合酶效率。证明了从粗PCR产物中成功进行基因分型。该方法可应用于任何实验室；它廉价、快速、简单，并且有可能被整合到DNA芯片形式中用于高通量焦磷酸测序分析。
