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Bulletin of Botanical Research ›› 2007, Vol. 27 ›› Issue (1): 77-81.doi: 10.7525/j.issn.1673-5102.2007.01.015

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Study on Optimization of ISSR Reaction Conditions for Coptis Chinensis Franch.

CHEN Da-Xia;LI Long-Yun;LU Cheng;ZHONG Guo-Yue;QU Xian-You;PENG Rui   

  1. (1.Chongqing Academy of Chinese Materia Medica,Chongqing 400065) (2.The Key Sericultural Laboratory of Southwest Agricultural University,Chongqing 400716)
  • Received:1900-01-01 Revised:1900-01-01 Online:2007-01-20 Published:2007-01-20
  • Contact: CHEN Da-Xia
  • Supported by:

Abstract: Based on the genomic DNA extracted from Coptis Chinensis Franch., this paper presented the effect of the main reaction system elements(Mg2+、dNTP、primer、template DNA、Taq DNA polymerase) and hot-cycle parameter(annealing temperature、cycles、denaturing time、annealing time and extension time) on ISSR-PCR which were tested by single or dual factor experiment, respectively to determine its optimal levels. A reaction system and amplified procedure suitable for Coptis Chinensis Franch were established,that is, 25μL amplification reactions system containing 1×PCRbuffer、1.5mmol·L-1 Mg2+、200μmol·L-1 dNTP、0.3μmol·L-1 primer、40 ng template DNA、1 U Taq DNA polymerase.The optimal amplified procedure was as follows: after a pre-denaturing of 30s at 94℃, 35 cycles were performed with denaturing of 30s at 94℃, annealing of 1min due to denaturing temperature of different primer, extension of 1.5min at 72℃, a final extension step of 7 min at 72℃ and hold at -4℃. This optimal system laid the standardization program of the identification of Coptis Chinensis Franch. and the efficient use of its germplasm resource.

Key words: Coptis Chinensis Franch., ISSR, reaction system, hot-cycle parameter

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