A bacterial artificial chromosome （BAC） library is an invaluable resource tool to initiate tea plant genomics research, and the preparation of high molecular weight （HMW） genomic DNA is a crucial first step for co...A bacterial artificial chromosome （BAC） library is an invaluable resource tool to initiate tea plant genomics research, and the preparation of high molecular weight （HMW） genomic DNA is a crucial first step for constructing a BAC Library. In order to construct a BAC library for enhancing tea plant genomics research, a new method for the preparation of tea pant high molecular weight （HMW） genomic DNA must be developed due to young tea plant leaves and shoots are notably rich in both tea polyphenols and tea polysaccharides. In this paper, a modified method for preparing high quality tea plant HMW genomi~ DNA was optimized, and the quality of tea plant genomic DNA was evaluated. The results were as follows： Critical indicators of HMW DNA preparation were the appearance of the smooth nuclei in solution （as opposed to sticky-gummy） before agarose plug solidification, non-dark colored nuclei plugs after lysis with an SDS/proteinase K solution, and the quality and quantity of HMW DNA fragments after restriction enzyme digestion. Importantly, 1% dissolved PVP-40 and 1% un-dissolved PVP-40 during the nuclei extraction steps, in conjunction with the removal of PVP-40 from the plug washing and nuclei lysis steps, were critical for achieving HWM tea plant DNA suitable for BAC library construction. Additionally, a third PFGE fraction selection step to eliminate contaminating small DNA fragments. The modifications provided parameters that may have prevented deleterious interactions from tea polyphenols and tea polysaccharides. The HMW genomic DNA produced by this new modified method has been used to successfully construct a large-insert tea plant BAC library, and thus may be suitable for BAC library construction from other plant species that contain similarly interfering compounds.展开更多
文摘A bacterial artificial chromosome （BAC） library is an invaluable resource tool to initiate tea plant genomics research, and the preparation of high molecular weight （HMW） genomic DNA is a crucial first step for constructing a BAC Library. In order to construct a BAC library for enhancing tea plant genomics research, a new method for the preparation of tea pant high molecular weight （HMW） genomic DNA must be developed due to young tea plant leaves and shoots are notably rich in both tea polyphenols and tea polysaccharides. In this paper, a modified method for preparing high quality tea plant HMW genomi~ DNA was optimized, and the quality of tea plant genomic DNA was evaluated. The results were as follows： Critical indicators of HMW DNA preparation were the appearance of the smooth nuclei in solution （as opposed to sticky-gummy） before agarose plug solidification, non-dark colored nuclei plugs after lysis with an SDS/proteinase K solution, and the quality and quantity of HMW DNA fragments after restriction enzyme digestion. Importantly, 1% dissolved PVP-40 and 1% un-dissolved PVP-40 during the nuclei extraction steps, in conjunction with the removal of PVP-40 from the plug washing and nuclei lysis steps, were critical for achieving HWM tea plant DNA suitable for BAC library construction. Additionally, a third PFGE fraction selection step to eliminate contaminating small DNA fragments. The modifications provided parameters that may have prevented deleterious interactions from tea polyphenols and tea polysaccharides. The HMW genomic DNA produced by this new modified method has been used to successfully construct a large-insert tea plant BAC library, and thus may be suitable for BAC library construction from other plant species that contain similarly interfering compounds.
