title: Preparation of single rice chromosome for construction of a DNA library using a laser microbeam trap.
abstract: We report the development of a laser micromanipulation system and its application in the isolation of individual rice chromosomes directly from a metaphase cell. Microdissection and flow sorting are two major methods for the isolation of single chromosome. These methods are dependent on the techniques of chromosome spread and chromosome suspension, respectively. In the development of this system, we avoided using chromosome spread and cell suspension was used instead. The cell wall of metaphase rice cell was cut by optical scissors. The released single chromosome was captured by an optical trap and transported to an area without cell debris. The isolated single chromosome was then collected and specific library was constructed by linker adaptor PCR. The average insert size of the library was about 300 bp. Two hundred inserts of chromosome 4 library were sequenced, and 96.5% were aligned to the corresponding sequences of rice chromosome 4. These results suggest the possible application of this method for the preparation of other subcellular structures and for the cloning of single macromolecule through a laser microbeam trap.
authors.affiliation: National Center for Gene Research, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200233, China.
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Liu
authors.forename: Haowei
authors.initials: H
authors.lastname: Wang
authors.forename: Yinmei
authors.initials: Y
authors.lastname: Li
authors.forename: Yesheng
authors.initials: Y
authors.lastname: Tang
authors.forename: Yilei
authors.initials: Y
authors.lastname: Liu
authors.forename: Xin
authors.initials: X
authors.lastname: Hu
authors.forename: Peixin
authors.initials: P
authors.lastname: Jia
authors.forename: Kai
authors.initials: K
authors.lastname: Ying
authors.forename: Qi
authors.initials: Q
authors.lastname: Feng
authors.forename: Jianping
authors.initials: J
authors.lastname: Guan
authors.forename: Chaoqing
authors.initials: C
authors.lastname: Jin
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Liren
authors.initials: L
authors.lastname: Lou
authors.forename: Zhuan
authors.initials: Z
authors.lastname: Zhou
authors.forename: Bin
authors.initials: B
authors.lastname: Han
mesh_terms: D032461:Chromosomes, Plant; D003001:Cloning, Molecular / Q000379:methods*; D018744:DNA, Plant; D015723:Gene Library; D008677:Metaphase; D042282:Microdissection; D012275:Oryza / Q000235:genetics*; D016133:Polymerase Chain Reaction
chemical_list: D018744:DNA, Plant