大白菜单拷贝序列的长片段PCR体系优化

Optimization of Long-range PCR System Based on Single Copy Sequences of Chinese Cabbage

目的序列长片段PCR产物可作为FISH技术的有效探针进行分子细胞遗传学研究。然而,传统PCR技术对于5 kb以上的长片段进行有效扩增很难。合适的反应条件及反应体系是进行长片段PCR有效扩增的必要前提。为了获得目的序列长片段PCR产物以用于FISH研究,根据大白菜A03染色体顶端无重复序列区段设计了80对长片段PCR引物,从基因组DNA模板质量、d NTPs浓度以及退火温度和延伸时间方面对PCR技术体系进行了优化。试验证明,选用幼苗嫩叶的基因组DNA和LA Taq DNA聚合酶可以提高长片段PCR引物的扩增质量和扩增效率;确定了适合5～15 kb长片段PCR的反应体系为20μL：50 ng/μL模板DNA 2μL,2.5 mmol/L d NTPs 1.6μL,10μmol/μL正反引物各1μL,10×LA PCR BufferⅡ（含Mg^2＋）2μL,5 U/μL LA Taq酶0.2μL;反应条件为98℃变性15 s;58～64℃退火10 s,68℃延伸5 min,35个循环;68℃延伸10 min,4℃保存。在大白菜基因组中成功获得了60对5～15 kb的扩增片段。为在大白菜粗线期染色体上开展长片段PCR-FISH技术研究及在近缘种间开展比较染色体涂染揭示进化关系奠定了理论基础。

The products obtained by long range PCR based on objective sequences were the effective probes for FISH to carry out molecular cytogenetic study. However,it is very difficult to amplify more than 5 kb-long fragments effectively by traditional PCR techniques. Suitable reaction conditions and reaction system are the necessary prerequisite for running effective LR-PCR amplification. In order to obtain LR-PCR products based on the objective sequences as FISH probes,80 pairs of LR-PCR primers were designed based on the sequences of repeat-free regions on the top of Chinese cabbage chromosome A03,and PCR technique system was optimized from the following aspects containing the template quality of Chinese cabbage genomic DNA,d NTPs concentration,annealing temperature and extended time. Results indicated that genomic DNA of seedling leaves and LA Taq polymerase enzyme could improve the amplification quality and efficiency of long-range PCR. And 5-15 kb PCR products could be amplified by the reaction system with 20 μL of total volume,comprising of 50 ng/μL DNA 2 μL,2. 5 mmol/L d NTPs 1. 6μL,10 μmol/μL of each primer 1 μL,10 × LA PCR Buffer Ⅱ（ including Mg^2＋） 2 μL,5 U/μL LA Taq enzyme0. 2 μL. PCR reaction procedure was followed as： 98 ℃ for 15 s; 58-64 ℃ for 10 s,68 ℃ for 5 min,35 cycles; final extension at 68 ℃ for 10 min kept at 4 ℃. Under these conditions,60 fragments with 5-15 kb length were obtained from Chinese cabbage genome. This research would establish the basis for carrying out long range PCR-FISHon meiotic pachytene chromosomes of Chinese cabbage and for clarifying evolutionary by comparing chromosome painting among close related species.