一种适合扩增枣基因片段侧翼序列的SON-PCR技术

摘要

[目的]建立一种适合枣相关基因侧翼序列延伸的SON-PCR技术.[方法]在原有SON-PCR基础上进行改进,将原有SON-PCR第2轮两段式反应改进为两步式,即第2轮首先加入单引物进行单侧嵌套引物引发5'端进行选择扩增,然后以第1步产物为模板,以少量的第1轮引物和第2轮引物进行特异性扩增,使特异性和效率进一步加强;将原来第2轮扩增模板浓度调整至10 ng·μL(-1)左右,以适合枣相关基因侧翼序列扩增.[结果]利用该体系对'骏枣'1条626 bp的片段进行了侧翼序列扩增,获得了1条934 bp的编码富含亮氨酸重复序列的相关片段;同时以其它2条枣基因片段为起始序列进行了侧翼序列扩增,进一步验证了此技术的通用性.[结论]建立了一种能够快速、高效地扩增枣基因片段侧翼序列的SON-PCR技术.%[Objective]In this experiment, Junzao was used as materials based on previous studies, to establish a SON-PCR technique for isolation unknown flanking sequence of Chinese jujube.[Method]The original two-stage second round of the SON-PCR reaction was improved to two-step: firstly, the PCR results of primary reaction were used as template, single nested primer was used to drive the 5’selected linear amplification; then, a small amount of the primer designed for primary reaction and primer designed for secondary reaction was added to specially amplificating secondary reaction.The specificity and efficiency were further enhanced, and the dilution of the original secondary reaction PCR template was adjusted to 10ng·μL-1 of the primary reaction result.[Result]Finally, the test data using the system to a 626bp fragment of 3 ‘flanking sequences were amplified to obtain a 934bp encoding leucine-rich repeat sequence related fragment.The universality of this technique was proved by using two other fragments.[Conclusion]The method can efficiently amplify unknown sequence flanking sequence.