HRCA技术在转基因植物检测中的应用

超分支滚环扩增技术（Hyperbranched rolling cycle amplification, HRCA）在近几年中逐渐引起人们的注意，并越来越多的用于基础研究和实际检测中。在本文中我们对该技术在转基因植物检测中的应用情况作了较全面的探索；根据4种转基因植物中常用的外源基因或DNA片段设计了4条锁式探针（Padlock probe），利用质粒pKK2328中的一段序列作为锁式探针中的共同连接部分，并根据该共同的连接部分序列设计一对通用的HRCA引物；利用同位素标记的锁式探针对HRCA反应中的连接一步的特异性研究表明，只有当锁式探针和相应的检测靶DNA同时存在于连接体系中时，锁式探针才能被有效地进行连接，从线性分子变为环型分子，在没有相应靶DNA存在时锁式探针仅以线性形式存在；连接时间的研究表明，如果所检测的靶DNA是质粒或较短的DNA片段时，较短的连接时间（5～10min）就可以取得理想的最终检测效果,如果检测的靶DNA是复杂的植物基因组DNA时，连接时间需要较大程度的延长（30～60min）才能取得理想的最终检测结果；HRCA的反应时间研究表明,较长的反应时间可以明显增加最终产物的量；对Bst DNA聚合酶大片段酶用量的研究表明，在其它条件不变的情况下酶的用量可以在较大的范围内变化（0.5u～4u）而不影响最终检测结果；在上述研究的基础上，对转基因烟草进行实际检测，取得了与预期一致的理想结果。为了提高检测效率，仿效复合式PCR（Multiplex PCR，MPCR）的原理采用复合式HRCA（Multiplex HRCA, MHRCA）方法对转基因烟草进行检测，并利用反向点杂交进行结果分析，取得同预期完全一致的结果。我们的研究表明HRCA方法完全可以用于转基因植物的检测，而且其使用比MPCR技术更方便，效率更高。

HRCA and Application in Detection of Genetically Modified Plant

In this article primary studies of the application of hyperbranched rolling cycle amplification (HRCA) in exogenous genes detection of transgenic plants were done. Four padlock probes were designed according to the sequences of four genes/DNA fragments that are used widely in transgenic plants; part of the sequence of pKK233 was chosen as the linking part of padlock probes and a pair of HRCA primers was designed according to the sequence of linking part. Study of the specificity of ligation in HRCA with isotope labeled padlock probes indicated padlock probes could be ringed effectively only when corresponding target DNA exited in the same reaction system and could not be ringed when there was no corresponding target DNA exited. Ligation time is very different according to the characteristic of target DNA being used. 5 min to 10 min is enough if the target DNA is plasmid; 30 min to 60 min is needed if the target is genome DNA of plant because it's sequence is more complex than that of plasmid's. HRCA time was analyzed which indicated longer reaction time can obviously increase the amount of products. Quantity of enzyme in HRCA was also analyzed. Different amount of enzyme (from 0.5 unit to 4 units) can give similar result when other conditions are not changed. On the basis of the research, transgenic tobacco was detected with these four padlock probes and the results were just as prospective. In order to increase the efficiency of detection, multiplex HRCA (MHRCA)was used. In MHRCA more than one padlock probes are used at the same time in the same reaction system to detect more than one targets. Because the amplification products of MHRCA will be complex and it is almost impossible to analyze with electrophoresis, so reverse blot is used. Detection results of transgenic tobacco with this method are the same with anticipation. Compare to MPCR method we established before MHRCA is more convenient to operate and more effective in detecting exogenous genes in transgenic plants.