中国听力语言康复科学杂志Issue(6)：418-423,6.DOI:10.3969/j.issn.1672-4933.2014.06.006
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
摘要
Abstract
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.关键词
GJB2基因/序列长度/长链PCR/琼脂糖凝胶电泳Key words
GJB2 gene/Sequence length/Long-chain PCR method/Agarose gel electrophoresis引用本文复制引用
王辉兵,于飞,戴朴,单希征,袁永一,张昕,康东洋,韩东一..GJB2全序列长链PCR和琼脂糖凝胶电泳方法研究[J].中国听力语言康复科学杂志,2014,(6):418-423,6.基金项目
国家自然科学基金面上项目（81070792）；武警总医院课题 （）
