| 摘要: |
| [目的]对限制性位点扩增多态性(Restriction site amplified polymorphism,RSAP)标记技术进行改进,并优化了其反应体系.[方法]遵循引物设计原则,使限制性位点序列位于中间.在其3'端增加3个选择性碱基,5'端设计为10～12个碱基的随机序列,设计了14条长为19 bp的限制性位点扩增多态性(RSAP)新引物.以大白菜、紫菜薹自交系及其F1、F2的DNA为模板,对新引物的反应体系进行了优化,并对其重复性和适用性进行了检验.＃[结果]新设计引物PCR扩增的前5个循环退火温度为35℃,后35个循环为52℃;在25 μL优化反应体系中,模板DNA用量为2.0μL(20.0 ng/μL)、Mg2+3.0μL(25 mmol/L)、Taq酶(5 U/μL)1.5 U、dNTPs 2.0 μL(2.5 mmol/L)、引物各0.6μL(10 μmol/L);新引物扩增出的谱带较原引物更多,多态性更好;只改变选择性碱基,新设计引物就能扩增出新的谱带;新设计引物的重复性、稳定性好,在养麦和小麦品系,以及紫菜薹、大白菜及其F1、F2中,新引物均能扩增出清晰谱带.[结论]新引物适用性和重复性好,可广泛应用于其他作物的基因组DNA分析. |
| 关键词: 限制性位点扩增多态性 引物设计 反应体系 体系优化 |
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| 基金项目:西北农林科技大学科研专项基金，国家高技术研究发展计划(863计划)? |
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| New primers design and reaction system optimization of restriction site amplified polymorphism (RSAP) marker technique |
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| Abstract: |
| 【Objective】 RSAP marker technique was improved and its reaction system was optimized.【Method】 The sequence of restriction site (4-6 bases) located in the middle,three selective nucleotides were added to its 3′end and 10 to 12 bases long of random sequence lied in its 5′end.Fourteen new primers of restriction site amplified polymorphism (RSAP) were redesigned 19 nucleotides long.Then,using DNA from inbreed lines’young leaves of Chinese cabbage (Brassica campestris L.ssp.Pekinensis), Purple caitai（Brassica compestris L.var.purpurea Bailey）and their F1,F2 as trial materials,its reaction systm was optimized and reproducibility and applicabtivity were tested. 【Result】 PCR amplification of new primers was run for the first 5 cycles with an annealing temperature of 35 ℃,followed by 35 cycles with an annealing temperature of 52 ℃;the optimum PCR reaction system of 25 μL included 2.0 μL DNA templates (20.0 ng/μL),3.0 μL of Mg2+ (25 mmol/L),1.5 U of Taq-DNA polymerase,2.0 μL of dNTPs (2.5 mmol/L) and 0.6 μL of each primers (10 μmol/L).New primers could amplify more strips and polymorphism was better than former primers.In the varieties of buckwheat and wheat,new primer could amplify vivid strips too.【Conclusion】 The applicability and reproductivity of new primers were very good,and application would be broad. |
| Key words: restriction site amplified polymorphism primer design reaction system optimization |
