GJB2全序列长链PCR和琼脂糖凝胶电泳方法研究

Long-chain PCR Method and Agarose Gel Electrophoresis for Full Sequence of GJB2 Gene

目的：探讨GJB2全序列长链PCR方法和琼脂糖凝胶电泳方法，以及影响长链PCR和电泳结果的可能因素。方法应用Primer Premier 5.0软件和Oligo 6 Demo软件针对GJB2全序列设计引物，应用DNA聚合酶KOD FX Neo试剂盒进行两步法长链PCR扩增，调整加入DNA模板量、PCR延伸时间、循环次数等影响PCR产物量，通过0.8%琼脂糖凝胶电泳检测PCR产物长度和量，调整加样槽的宽度、加样量、电泳电压、电流、电泳时间得到清晰条带，若PCR产物存在大片段碱基插入或缺失，用限制性内切酶BamHI进行内切酶反应，初步判断插入或缺失的大致位置。结果正向引物F：5’-AGATCGGGACCTCGAAGGGGACTTG-3’；反向引物R：5’-AGGTGGGCACGGGGTTAGGTAGAAA-3’，扩增片段长5887 bp。长链PCR条件为：50μl的反应体系中加入2μl（约40 ng）的基因组DNA，预变性94℃2分钟，变性98℃10秒，68℃延伸5分钟，共32个循环。电泳条件为：加样槽5 mm宽，每槽加样0.8μl PCR产物，电泳电压50 V，电流50 mA，电泳时间140分钟。结论应用DNA聚合酶KOD FX Neo试剂盒进行两步法长链PCR，可进行GJB2全序列扩增，影响PCR的可能因素为引物、DNA模板的质和量、延伸时间、循环次数等。0.8%琼脂糖凝胶电泳可获得较好的分离效果，影响电泳可能的因素为加样槽宽度、加样量、电泳电压、电流、电泳时间等。

Objective To explore the long-chain PCR method and agarose gel electrophoresis for the full sequence of GJB2 gene and to discuss the possible factors affecting the long-chain PCR and electrophoresis results. Methods The primers for the full sequence of GJB2 gene were designed by Primer Premier 5.0 software and Oligo 6 Demo software and the sequence were amplified using DNA polymerase of KOD FX Neo kit by the two-step PCR method. The product amount of PCR was controlled by the amount of the DNA template, the extension time and the cycles of PCR, and so on. The sequence length of PCR product was detected by 0.8%agarose gel electrophoresis. To obtain a clear electrophoresis strip, we changed the width of the well, the volume of sample of PCR product, the electrophoresis voltage and current, electrophoresis time, and so on. If obvious increasing or decreasing of the sequence length of PCR products had been detected, there should exist insertion or deletion of large fragment nucleotide in the GJB2 gene sequence. We determined the approximate location of the insertion or deletion by restriction enzyme reaction of BamHI enzyme. Results The forward primer was 5＇-AGATCGGGACCTCGAAGGGGACTTG-3＇ and the reverse primer was 5＇-AGGTGGGCACGGGGTTAGGTAGAAA -3＇, and the length of the amplified fragment was 5887 bp. The optimal condition of long-chain PCR for the full sequence of the GJB2 gene was as following： the volume of genomic DNA was 2 μl（about 40ng DNA） in a reaction system of 50 μl, pre-denaturation at 94℃for 2 minutes, denaturation at 98℃for 10 seconds, extension at 68℃for 5 minutes, a total of 32 cycles. The condition of 0.8% agarose gel electrophoresis was as following： the width of the well was 5 mm, the total volume of sample was 6 μl, the volume of PCR product was 0.8 μl, the volume of the 1 kb DNA ladder was 0.8 μl, the electrophoresis voltage was 50 Volts, the current intensity was 50 mA, the electrophoresis time was about 140 minutes. Conclusion The full sequence of GJB2 gene could be amplified by the two-step long chain PCR method using DNA polymerase of KOD FX Neo kit and be separated by 0.8%agarose gel electrophoresis.