Optimization of SSR-PCR Reaction System in Eucommiae Cortex

MI Ya-nan; ZHANG Shui-han; CAI Yuan; LIANG Xue-juan; JIANG Chao

doi:10.13422/j.cnki.syfjx.2015020001

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Optimization of SSR-PCR Reaction System in Eucommiae Cortex

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- Optimization of SSR-PCR Reaction System in Eucommiae Cortex
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 21, Issue 2, Pages: 1-6(2015)
- 作者机构：
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.2015020001
  CLC： S567.19
- Published：2015
- 稿件说明：
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MI Ya-nan, ZHANG Shui-han, CAI Yuan, et al. Optimization of SSR-PCR Reaction System in Eucommiae Cortex[J]. Chinese journal of experimental traditional medical formulae, 2015, 21(2): 1-6.
DOI：

MI Ya-nan, ZHANG Shui-han, CAI Yuan, et al. Optimization of SSR-PCR Reaction System in Eucommiae Cortex[J]. Chinese journal of experimental traditional medical formulae, 2015, 21(2): 1-6. DOI： 10.13422/j.cnki.syfjx.2015020001.

摘要

目的: 建立杜仲简单重复序列-聚合酶链式反应(SSR-PCR)的优化反应体系

为杜仲种质间的遗传差异及亲缘关系分析提供参考。方法: 选择Taq DNA聚合酶

模板DNA

Mg2+

引物及dNTP浓度为考察因素

利用单因素试验和L16(45)正交试验优选杜仲的SSR-PCR 反应体系

运用SPSS软件对试验结果进行分析。结果: 各因素水平变化对反应体系影响顺序依次为Mg2+>Taq DNA聚合酶>引物>dNTP>模板DNA。优化的PCR反应体系为10×PCR缓冲液2 μL

Taq DNA聚合酶0.5 U

Mg2+浓度1.25 mmol ·L-1

dNTP浓度0.2 mmol ·L-1

引物浓度0.3 μmol ·L-1

模板DNA用量60 ng

定容至20 μL。结论: 利用该体系进行扩增

所得谱带明亮清晰

稳定性和重复性良好

适用于杜仲不同居群间亲缘关系和遗传多样性分析。

Abstract

Objective: To optimize simple sequence repeat-polymerase chain reaction(SSR-PCR) system for providing a reference to genetic diversity and phylogenetic analysis among germplasms of Eucommiae Cortex. Method: Orthogonal design and single factor tests were adopted to optimize SSR-PCR system of Eucommiae Cortex with Taq DNA polymerase

template DNA

Mg2+

primers and dNTP concentration as factors

test data were analyzed by SPSS software. Result: Effects of each factors were in the order of Mg2+>Taq DNA polymerase>primers>dNTP>DNA template.Optimal reaction system was as follows:10×PCR buffer of 2 μL

Taq DNA polymerase of 0.5 U

Mg2+ of 1.25 mmol · L-1

dNTP concentration of 0.2 mmol · L-1

primers of 0.3 μmol · L-1 and DNA template of 60 ng in 20 μL reaction solution. Conclusion: Bands are bright and clear with good stability and reproducibility by amplification of this optimized SSR-PCR system

it is suitable for genetic diversity and phylogenetic analysis among germplasms of Eucommiae Cortex.

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Related Author

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Related Institution

National Resource Center for Chinese Materia Medica，China Academy of Chinese Medical Sciences

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