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Citation:

Optim iza tion for SSR-anchored PCR Reaction Systemin Endangered Plant Taxus yunnanensis

  • Received Date: 2007-05-16
  • The interaction among different factorswas usually ignored during experimental design with orthogonal design which waswidely used in op timizing for PCR reaction system. Orthogonal design L64 (421 ) involving interactionwas used to op timize simp le sequence repeated-anchored PCR by degenerate p rimer ( SSRanchored PCR) amp lification system on T. yunnanensis in five factors: Taq DNA polymerase, Mg2+ , dNTP,DNA and p rimer at four levels.The analytic results by software SAS showed that: ①Three factors (Taq DNA polymerase, Mg2+ , dNTP) and the twointeractions ( Taq ×Mg2+ , Mg2+ ×dNTP) had notable effect on the SSR2anchored PCR amp lification system; ②Anop timal SSR-anchored PCR reaction system was established containing 1 ×PCR buffer, 4 U Taq DNA polymerase,2. 0 mmol·L-1Mg2+ , 0. 4 mmol·L-1 dNTP, 75 ngDNA, 1. 0μmol·L-1 p rimer in a total volume of 20μL reac2tion system. Moreover the op timal annealing temperature (54 ℃) was p roposed by gradient PCR. Orthogonal designinvolving interaction is of special app lication value as it can not only screen out the best PCR reaction system quicklyand effectively, but also reveal the extent to which the testing factors and their interaction can affect the PCR amp lification, and thusmake the analytic resultmore objective and accurate. While, further research of the judgmentalstandard to the electrophoretogram of PCR amp lification p roducts is still needed to imp rove both the facility of themethod and the reliability of experimental data.
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Optim iza tion for SSR-anchored PCR Reaction Systemin Endangered Plant Taxus yunnanensis

  • 1. Research Institute of Resource Insects, CAF, Kunming 650224, Yunnan, China

Abstract: The interaction among different factorswas usually ignored during experimental design with orthogonal design which waswidely used in op timizing for PCR reaction system. Orthogonal design L64 (421 ) involving interactionwas used to op timize simp le sequence repeated-anchored PCR by degenerate p rimer ( SSRanchored PCR) amp lification system on T. yunnanensis in five factors: Taq DNA polymerase, Mg2+ , dNTP,DNA and p rimer at four levels.The analytic results by software SAS showed that: ①Three factors (Taq DNA polymerase, Mg2+ , dNTP) and the twointeractions ( Taq ×Mg2+ , Mg2+ ×dNTP) had notable effect on the SSR2anchored PCR amp lification system; ②Anop timal SSR-anchored PCR reaction system was established containing 1 ×PCR buffer, 4 U Taq DNA polymerase,2. 0 mmol·L-1Mg2+ , 0. 4 mmol·L-1 dNTP, 75 ngDNA, 1. 0μmol·L-1 p rimer in a total volume of 20μL reac2tion system. Moreover the op timal annealing temperature (54 ℃) was p roposed by gradient PCR. Orthogonal designinvolving interaction is of special app lication value as it can not only screen out the best PCR reaction system quicklyand effectively, but also reveal the extent to which the testing factors and their interaction can affect the PCR amp lification, and thusmake the analytic resultmore objective and accurate. While, further research of the judgmentalstandard to the electrophoretogram of PCR amp lification p roducts is still needed to imp rove both the facility of themethod and the reliability of experimental data.

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