摘要
以牡丹基因组DNA为模板,采用L16(45)正交试验设计对影响目标起始密码子多态性-聚合酶链式反应(SCoT-PCR)的五因素(Taq酶用量、Mg2+浓度、模板DNA用量、dNTPs浓度和引物浓度)进行优化试验,建立了优化的牡丹SCoT-PCR反应体系:Mg2+2.50 mmol/L、dNTPs 0.25 mmol/L、引物0.60μmol/L、Taq DNA聚合酶0.50 U、模板DNA 1.00 ng/μL,1×PCR-Buffer,总体积20.00μL。比较各因素对扩增反应的结果,其中以Mg2+浓度的影响最大,DNA模板用量的影响最小。运用牡丹17个品种验证了该体系稳定可靠,并从36个SCoT引物中筛选出扩增条带清晰、多态性丰富的24个引物。这一体系的建立及多态性引物的筛选为今后利用SCoT标记技术对牡丹进行相关研究提供了科学依据。
The orthogonal design table of LI6 （4.5 ） was used to optimize the five factors （ Taq DNA polymerase, Mg2＋ , DNA template, dNTPs and primer concentrations）of SCoT-PCR（ start codon targeted polymorphism）system in tree peony. The results showed that the optimized system was as follows： a total volume of 20 μL including 1.0 ng/μL DNA template,0.50 U Taq polymerase,0.60 μmol/L primer,2.50 mmol/L Mg2＋ ,0.25 mmol/L dNTPs and 1 PCR Buffer. Each factor had different effect on the results. The concentration of Mg2＋ was the key factor affecting the SCoT-PCR system. The optimized SCoT-PCR system was tested on seventeen peony genus,and the result was stable and reliable. From the 36 primer combinations tested, Twenty-four were selected with clear band patterns and abundant polymorphism. The optimized SCoT-PCR system and polymorphism primmer combinations provided the basis for further research on Paeonia suffruticosa.
出处
《华北农学报》
CSCD
北大核心
2011年第5期92-96,共5页
Acta Agriculturae Boreali-Sinica
基金
国家自然科学基金(31070620)
河南省重大科技攻关项目(091100110100)
河南省重点科技攻关项目(092102110024)
洛阳市科技攻关项目(0802017A)
