Ponelies N, Stein N, Weber G
Institute of Plant Breeding, Seed Science and Population Genetics, University of Hohenheim, D-70593 Stuttgart, Germany.
Nucleic Acids Res. 1997 Sep 1;25(17):3555-7. doi: 10.1093/nar/25.17.3555.
An improved method was developed for microdissection and cloning of metaphase as well as pachytene chromosomes. The protocol incorporates efficient ligation of chromosomal DNA with linker adaptors, abolishment of microcloning steps and the reduction of micromanipulation. The threshold for amplifying genomic DNA template was in the range of 2-20 femtogram. The amplification products had a size distribution between 200 and 1300 bp (average 500 bp). Using pachytene chromosomes of maize the selectivity for segment-specific libraries can be increased between 10- and 20-fold. The approach described here is being applied to the fine mapping and isolation of genes conveying resistance against plant pathogens.
开发了一种用于中期和粗线期染色体显微切割及克隆的改进方法。该方案包括将染色体DNA与接头衔接子进行高效连接、取消微克隆步骤以及减少显微操作。扩增基因组DNA模板的阈值在2至20飞克范围内。扩增产物的大小分布在200至1300碱基对之间（平均500碱基对）。使用玉米粗线期染色体，片段特异性文库的选择性可提高10至20倍。此处所述方法正应用于对植物病原体抗性基因的精细定位和分离。
