【摘要】 研究旨在鉴定两条在非变性聚丙烯酰胺凝胶电泳中总是伴随杂合子出现的非目的条带。实验切胶回收了目的条带和非目的条带,以此为模板进行第二次PCR扩增,然后将PCR产物按不同组合进行混合并施加不同处理再次进行电泳,另外,人工合成了四条包含该缺失位点的寡核苷酸单链,对条带形成的过程进行了模拟。结果表明以两条非目的条带为模板的第2次PCR产物的电泳结果均有4条带,与杂合子样本4条带的迁移率完全一致,以2条目的条带为模板的第2次PCR产物电泳条带的迁移率分别与其本身保持一致;将以2条目的条带为模板的第2次PCR产物混合并经过变性复性后,其结果出现非目的条带和目的条带,也与杂合子样本4条带的迁移率完全一致,而如果只是简单混合并未经过变性复性处理,其电泳结果只出现目的条带。另外,在利用寡核苷酸单链进行模拟的实验中发现,异源双链迁移率较低,形成了非目的条带,而正常双链迁移率较高,形成目的条带。结果提示,这种与杂合子相伴产生的非目的条带,并不是非特异性扩增的结果,而是由于模板为缺失突变杂合子,在复性过程中,PCR产物中的一部分碱基缺失的正负单链会与不缺失的正负单链发生互补,而在发生碱基缺失的位置,会有突环产生,产生的突环进而导致杂合双链迁移率降低,最终形成了两条迁移率较低的非目的条带。更多还原

【Abstract】 This experiment was conducted to identify two nontargeted bands accompanied with heterozygote in nondenatured polyacrylamide gel electrophoresis. The nontargeted and targeted bands were cut and reclaimed, and used into PCR as templates. The PCR products were electrophoresised according to different combinations and disposals. In addition, 4 synthesized single strands were applied to simulate the formation process of nontargeted and targeted bands. The results showed that the mobility property of the PCR products of DNA extracted from untargeted bands was the same as that of the PCR products of genomic DNA from heterozygote. The mobility property of the PCR products of DNA extracted from either targeted band was the same as itself. If the PCR products of DNA extracted from the two targeted bands were mixed and treated with denaturation and renaturation in PCR instrument, the mobility property of them was also the same as that of the PCR products of genomic DNA from heterozygote; if mixed without other treatment, there were only two targeted bands appeared. In the simulation experiments, the untargeted bands were formed by the heteroduplex DNA strands and the targeted bands by the homoduplex DNA strands. These results indicated that the two nontargeted bands were not nonspecific amplicons. Because of a 3 bp deletion in one of two targeted double-stranded DNAs, two heteroduplex DNAs formed in the processes of denaturation and renaturation of PCR for random pairing of single-stranded DNAs, thus, convex rings were produced consequently. The convex rings decreased the mobility of the two heteroduplex DNAs and the two delayed heteroduplex DNAs formed the two nontargeted bands.更多还原