A protocol of simple rapid microdissection of single-chromosome, amplification and cloning of its DNA from Lilium regale Wilson is described. Single-chromosome, microdissected by micromanipulator, was put into a 0 5 m...A protocol of simple rapid microdissection of single-chromosome, amplification and cloning of its DNA from Lilium regale Wilson is described. Single-chromosome, microdissected by micromanipulator, was put into a 0 5 mL Eppendorf tube and digested with Sau3A, and then the Sau3A linker adaptors were ligated to the ends of DNA fragments. After 2 rounds of PCR amplification with one chain of linker adaptor as primer, the PCR products thus obtained have a length of 300-2 500 base pairs (bp) with predominant fragments at about 1 000 bp. Southern blot analysis confirmed that the PCR products originated from the genome of Lilium regale Wilson. By cloning the amplification products from the second round of PCR, single-chromosome DNA library was constructed, in which about as many as 100 000 recombinant clones were produced. A total number of 84 clones were analysed, and it was revealed that the inserts ranged in size from 300 to 1 800 bp, with an average of 780 bp. Compared with the methods described in other literature, this protocol, eliminating the need for enzymatic digestion and ligating micromanipulation of chromosomal DNA in nanoliter volumes, permits the efficient amplification of single chromosome (not tens of chromosomes as reported before) and the fragments (780 bp in average) cloned in this study are longer than those reported before (650 bp in average).展开更多
文摘A protocol of simple rapid microdissection of single-chromosome, amplification and cloning of its DNA from Lilium regale Wilson is described. Single-chromosome, microdissected by micromanipulator, was put into a 0 5 mL Eppendorf tube and digested with Sau3A, and then the Sau3A linker adaptors were ligated to the ends of DNA fragments. After 2 rounds of PCR amplification with one chain of linker adaptor as primer, the PCR products thus obtained have a length of 300-2 500 base pairs (bp) with predominant fragments at about 1 000 bp. Southern blot analysis confirmed that the PCR products originated from the genome of Lilium regale Wilson. By cloning the amplification products from the second round of PCR, single-chromosome DNA library was constructed, in which about as many as 100 000 recombinant clones were produced. A total number of 84 clones were analysed, and it was revealed that the inserts ranged in size from 300 to 1 800 bp, with an average of 780 bp. Compared with the methods described in other literature, this protocol, eliminating the need for enzymatic digestion and ligating micromanipulation of chromosomal DNA in nanoliter volumes, permits the efficient amplification of single chromosome (not tens of chromosomes as reported before) and the fragments (780 bp in average) cloned in this study are longer than those reported before (650 bp in average).
