基于果蝇DNA甲基化的AFLP体系的建立及优化

Establishment and Optimization of AFLP Analysis System for DNA Methylation in Drosophila

以黑腹果蝇(Drosophila melanogaster)Canton-S品系为材料,采用改良SDS法提取高质量DNA,对连接、预扩增及选择性扩增进行分析,建立适用于果蝇基因组DNA甲基化多态性研究的AFLP优化体系:1)10μL连接体系加T4连接酶1 U,AluⅠ接头50 pmol,EcoR Ⅰ接头5 pmol,4℃反应12 h;2)25μL预扩增体系含Mg2+0.2 mmol/L,dNTPs 0.15 mmol/L,模板1.0μL,Taq DNA聚合酶2 U,E-00 50 ng,A-00 50 ng;3)25μL选择性扩增体系含Mg2+0.1 mmol/L,dNTPs 0.15 mmol/L,模板2.0μL,Taq DNA酶1.5 U、E+340 ng,A+3 40 ng.该体系稳定性高、重复性好,适用于果蝇基因组DNA甲基化多态性研究.

High quality genomic DNA from Canton-S strain of Drosophila melanogaster was extracted by improved SDS method and several key factors,including EcoR Ⅰ/Alu Ⅰ digestion,adapter ligation,pre-amplification,selective amplification were analyzed.The optimized system was obtained as follows：1） ligation reaction system：T4 ligase 1 U,A-adapter 50 pmol,E-adapter 5 pmol,12 h for ligation at 4 ℃;2） Pre-amplification reaction was performed in a 25 μL volume containing Mg2＋ 0.2 mmol/L,dNTPs 0.15 mmol/L,E-00 50 ng,A-00 50 ng,2.5 μL of template DNA,Taq DNA polymerase 2 U;3） The selective amplification was contained Mg2＋ 0.1 mmol/L,dNTPs 0.15 mmol/L,2.0 μL of the diluted product,Taq DNA polymerase 1.5 U,E＋3 40 ng,A＋3 40 ng,and the total volume was 25 μL.The clear AFLP fingerprintings have been obtained.By virtue of high stability and good repeatability,this optimized AFLP system can be utilized for the methylation analysis in fruit fly.