Cloning and expression of a novel lipase gene from Pseudomonas fluorescens B52 |
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Authors: | Zhengbing Jiang Yitao Zheng Yu Luo Gang Wang Hongping Wang Yushu Ma Dongzhi Wei |
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Institution: | (1) The Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 10081 Beijing, China;(2) Life Science and Technology College of Shanghai Jiao Tong University, 200030 Shanghai, China |
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Abstract: | Here we describe an advanced polymerase chain reaction (PCR) technique, the compatible ends ligation inverse PCR (CELI-PCR)
for chromosome walking. In CELI-PCR, several restriction enzymes, which produce compatible cohesive ends, were used to digest
target DNA simultaneously or sequentially to produce DNA fragments of suitable size. DNA fragments were then easily circularized
and PCR amplification could be carried out efficiently. The previous limitations of inverse PCR were overcome, such as unavailable
restriction sites, poor template DNA circularization, and low amplification efficiency. Therefore, successive chromosome walking
was performed successfully. Our work, isolating a 11,395-bp fragment from Gossypium hirsutum, was presented as an example to describe how CELI-PCR was carried out. |
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Keywords: | Compatible ends ligation inverse PCR (CELI-PCR) chromosome walking Gossypium hirsutum |
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