Construction of single-chromosome DNA library fromLilium regale Wilson |
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Authors: | Benyuan Dang Zanmin Hu Yihua Zhou Lihua Cui Lanlan Wang Liangcai Li Zhenghua Chen |
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Institution: | (1) Institute of Genetics, Chinese Academy of Sciences, 100101 Beijing, China |
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Abstract: | A protocol of simple rapid microdissection of single-chromosome, amplification and cloning of its DNA fromLilium 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 300–2500 base pairs (bp) with
predominant fragments at about 1000 bp. Southern blot analysis confirmed that the PCR products originated from the genome
ofLilium 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 100000 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 1800 bp, with an average of780 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). |
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Keywords: | Lilium regale Wilson single-chromosome DNA library |
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