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中国农学通报

中国农学通报 ›› 2009, Vol. 25 ›› Issue (18): 41-46.

• 生物技术科学 • 上一篇    下一篇

均匀设计优化野生狗牙根的SRAP-PCR反应体系

朱红霞1,胡利宗2,邓小莉1   

  • 收稿日期:2009-04-30 修回日期:2009-05-21 出版日期:2009-09-20 发布日期:2009-09-20

Optimization of SRAP-PCR amplification system for wild cynodon dactylon by uniform design

  • Received:2009-04-30 Revised:2009-05-21 Online:2009-09-20 Published:2009-09-20

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摘要：

采用均匀设计方法，对影响野生狗牙根SRAP-PCR体系的MgCl2、dNTPs、引物、Taq DNA聚合酶和模板浓度等分别进行了U16(45 )和 U12(35 )两轮优化，建立了适用于野生狗牙根的SRAP-PCR反应体系。该优化的25ul反应体系包含MgCl2 3.5 mmol/L，dNTPs 0.20nmol/L，引物0.44umol/L，TaqDNA聚合酶1.50 U，模板28ng/L。在此基础上又对退火温度进行了摸索，结果发现扩增结果对退火温度变化不敏感。最后运用优化体系对来自不同地区的10份野生狗牙根的16个单株的DNA进行扩增验证，结果获得的DNA条带清晰，多态性比较丰富。说明该优化的SRAP-PCR体系可用于野生狗牙根不同种间亲缘关系、系统进化和遗传多样性等领域的研究。

关键词: 西部, 西部, 生态补偿机制, 可持续发展

Abstract:

Uniform design was applied to optimize SRAP(Sequence-related amplified polymorphism)-PCR(polymerase chain reaction) system of cynodon dactylon. Five main factors influenced on SRAP-PCR, including MgCl2, primer, dNTP, TaqDNA polymerase and DNA template were tested using uniform design U16(45 ) and U12(35 ). A 25 ul suitable SRAP-PCR system for cynodon dactylon was established. The SRAP-PCR amplification reaction system was consisted of 3.5 mmol/L MgCl2，0.20mmol/L dNTPs, 0.44umol/L primer,1.50U TaqDNA polymerase and 28g/L template. Annealing temperature was further researched and found that it has no significant influence on amplification results. By using this optimal system，the genomic DNA of other 10 samples of cynodon dactylon was amplified，and DNA bands with high polymorphism were clear. In conclusion, the optimization of SRAP-PCR amplification system can be used to study genetic relationship，systematic evolution and genetic diversity of cynodon dactylon.