Preparation of single rice chromosome for construction of a DNA library using a laser microbeam trap.
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.
Liu
Xiaohui
X
National Center for Gene Research, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200233, China.
Wang
Haowei
H
Li
Yinmei
Y
Tang
Yesheng
Y
Liu
Yilei
Y
Hu
Xin
X
Jia
Peixin
P
Ying
Kai
K
Feng
Qi
Q
Guan
Jianping
J
Jin
Chaoqing
C
Zhang
Lei
L
Lou
Liren
L
Zhou
Zhuan
Z
Han
Bin
B
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
D018744:DNA, Plant