[Objective] This study was conducted to optimize PCR-SSCP reaction system and conditions for hawthorn （Crataegus spp.）. [Method] The chloroplast DNA and nuclear DNA of hawthorn leaf were extracted with improved CTAB...[Objective] This study was conducted to optimize PCR-SSCP reaction system and conditions for hawthorn （Crataegus spp.）. [Method] The chloroplast DNA and nuclear DNA of hawthorn leaf were extracted with improved CTAB method. Pdmers for the PCR amplification of chloroplast DNA and nuclear DNA were select- ed from eight pairs of candidate pdmers, and the PCR-SSCP reaction system and reaction conditions were optimized. The PCR products were detected by agarose gel electrophoresis, and the denatured PCR-SSCP products were analyzed by native polyacrylamide gel. [Result] Five pairs primers （psbA-tmH, ropB, ropL, rpoC1 and ITS2） were proved to be suitable for PCR-SSCP in hawthorn, including four for chloroplast DNA and one for nuclear DNA. The clear electrophoretogram of PCR- SSCP in hawthorn was obtained by performing electrophoresis in 0.5×TBE buffer, at 4 ℃ and 200 V for 3-4 h, using 6% native polyacrylamide gel （crosslinking ratio at 29：1）, and the PCR product had been mixed with an equal volume of loading buffer containing 1% NaOH （without glycerol） and denatured at 98℃ for 15 min. [Conclu- sion] The results may lay the foundation of SSCP analysis of hawthom.展开更多
文摘[Objective] This study was conducted to optimize PCR-SSCP reaction system and conditions for hawthorn （Crataegus spp.）. [Method] The chloroplast DNA and nuclear DNA of hawthorn leaf were extracted with improved CTAB method. Pdmers for the PCR amplification of chloroplast DNA and nuclear DNA were select- ed from eight pairs of candidate pdmers, and the PCR-SSCP reaction system and reaction conditions were optimized. The PCR products were detected by agarose gel electrophoresis, and the denatured PCR-SSCP products were analyzed by native polyacrylamide gel. [Result] Five pairs primers （psbA-tmH, ropB, ropL, rpoC1 and ITS2） were proved to be suitable for PCR-SSCP in hawthorn, including four for chloroplast DNA and one for nuclear DNA. The clear electrophoretogram of PCR- SSCP in hawthorn was obtained by performing electrophoresis in 0.5×TBE buffer, at 4 ℃ and 200 V for 3-4 h, using 6% native polyacrylamide gel （crosslinking ratio at 29：1）, and the PCR product had been mixed with an equal volume of loading buffer containing 1% NaOH （without glycerol） and denatured at 98℃ for 15 min. [Conclu- sion] The results may lay the foundation of SSCP analysis of hawthom.
