Potato variety Kexin18 was used as testing materials in this research to study the influence on main components in multiplex PCR system, different primer ratios and annealing temperatures in SSR marker amplification. ...Potato variety Kexin18 was used as testing materials in this research to study the influence on main components in multiplex PCR system, different primer ratios and annealing temperatures in SSR marker amplification. Concentration and gradient experiments for four components （enzyme, MgCl2, DNA template and dNTPs） in PCR system were used in the research with the concentration of the other component remained the same; the orthogonal design L9 （34） was applied in the optimization of four sets of primers （STM0014, Pat, SSI, and UGP） in the reaction system at three levels; the temperature gradient selection was used to find out the optimum annealing temperature for the primer. The optimized multiplex PCR system of potato SSR marker with a total volume of 20 μL ： 2.5 μL 25 mmol.L-1 MgCl2, 0.6 μL 10 mmol·L-1 dNTPs, 0.8 U Taq, 80 ng DNA template was ultimately established through the comparison and analysis of test results; the ratio of four pairs of 4 mmol. L1 primers was 2 ： 1 ： 2 ： 3, and the annealing temperature was 54.7℃. The optimized reaction system could be repeated stably; and the stable and reliable amplification results were able to clearly distinguish different potato varieties. This research built the solid foundation for the further study of genetic diversity of potato germplasms and construction of DNA fingerprinting..展开更多
Taking China’s endemic tree species Alnus cremastogyne and other 5 Alnus species as test materials, the DNA extraction of genome and the amplification of PCR were studied. Two methods were tested, i.e. freezi...Taking China’s endemic tree species Alnus cremastogyne and other 5 Alnus species as test materials, the DNA extraction of genome and the amplification of PCR were studied. Two methods were tested, i.e. freezing treatment+5% CTAB and liquid nityrogen+5% CTAB. It is proved that the optimum PCR program is as follows: predenaturating under 94 ℃ for 3 min., denaturating under 94 ℃ for 18 seconds, annealing under 36 ℃ for 80 seconds and extending under 72 ℃ for 120 seconds. After 40 cycles, the sample is reacted for 10 minutes under 72 ℃. PCR system includes buffer 2.5 μL, dNTP 2.5 μL, primer (s60+s155) 2 mmol·L-1, Mg2+ 3.0 mmol·L-1, Tap enzyme 1 U, DNA 40 mg, then adding ddH2O to 20 μL. This study may provide some references for the application of RAPD technique in genetic research of alder tree species.展开更多
利用正交试验设计L9(34)的方法,最终建立了北重楼ISSR-PCR的优化反应体系,即20μL反应体系中包含1×buffer,dNTP 175μmol.L-1,Prim er 0.8μmol.L-1,Mg2+1.4 mmol.L-,Taq酶2.5 U及模板DNA 80 ng。适宜的扩增程序为95℃预变性5 m in...利用正交试验设计L9(34)的方法,最终建立了北重楼ISSR-PCR的优化反应体系,即20μL反应体系中包含1×buffer,dNTP 175μmol.L-1,Prim er 0.8μmol.L-1,Mg2+1.4 mmol.L-,Taq酶2.5 U及模板DNA 80 ng。适宜的扩增程序为95℃预变性5 m in;94℃变性40 sec,55℃退火45 sec,72℃延伸90 sec,40个循环;72℃延伸7 m in,4℃保存。该优化体系的建立,将为北重楼及其近缘种的系统学和种质资源鉴定及遗传多样性的研究奠定基础。展开更多
基金Supported by the International Cooperation Project of Heilongjiang Science and Technology Department (WC05B08)
文摘Potato variety Kexin18 was used as testing materials in this research to study the influence on main components in multiplex PCR system, different primer ratios and annealing temperatures in SSR marker amplification. Concentration and gradient experiments for four components （enzyme, MgCl2, DNA template and dNTPs） in PCR system were used in the research with the concentration of the other component remained the same; the orthogonal design L9 （34） was applied in the optimization of four sets of primers （STM0014, Pat, SSI, and UGP） in the reaction system at three levels; the temperature gradient selection was used to find out the optimum annealing temperature for the primer. The optimized multiplex PCR system of potato SSR marker with a total volume of 20 μL ： 2.5 μL 25 mmol.L-1 MgCl2, 0.6 μL 10 mmol·L-1 dNTPs, 0.8 U Taq, 80 ng DNA template was ultimately established through the comparison and analysis of test results; the ratio of four pairs of 4 mmol. L1 primers was 2 ： 1 ： 2 ： 3, and the annealing temperature was 54.7℃. The optimized reaction system could be repeated stably; and the stable and reliable amplification results were able to clearly distinguish different potato varieties. This research built the solid foundation for the further study of genetic diversity of potato germplasms and construction of DNA fingerprinting..
文摘Taking China’s endemic tree species Alnus cremastogyne and other 5 Alnus species as test materials, the DNA extraction of genome and the amplification of PCR were studied. Two methods were tested, i.e. freezing treatment+5% CTAB and liquid nityrogen+5% CTAB. It is proved that the optimum PCR program is as follows: predenaturating under 94 ℃ for 3 min., denaturating under 94 ℃ for 18 seconds, annealing under 36 ℃ for 80 seconds and extending under 72 ℃ for 120 seconds. After 40 cycles, the sample is reacted for 10 minutes under 72 ℃. PCR system includes buffer 2.5 μL, dNTP 2.5 μL, primer (s60+s155) 2 mmol·L-1, Mg2+ 3.0 mmol·L-1, Tap enzyme 1 U, DNA 40 mg, then adding ddH2O to 20 μL. This study may provide some references for the application of RAPD technique in genetic research of alder tree species.
文摘利用正交试验设计L9(34)的方法,最终建立了北重楼ISSR-PCR的优化反应体系,即20μL反应体系中包含1×buffer,dNTP 175μmol.L-1,Prim er 0.8μmol.L-1,Mg2+1.4 mmol.L-,Taq酶2.5 U及模板DNA 80 ng。适宜的扩增程序为95℃预变性5 m in;94℃变性40 sec,55℃退火45 sec,72℃延伸90 sec,40个循环;72℃延伸7 m in,4℃保存。该优化体系的建立,将为北重楼及其近缘种的系统学和种质资源鉴定及遗传多样性的研究奠定基础。
