关键词: 嵌套缺失技术, 影响因素, NGAL基因

Abstract: BACKGROUND ＆ AIM: To explore the influencing factors of nested deletions and find some methods to resolve interrelated problems. MATERIAL AND METHODS: Alkaline lysis, purification by polyethylene glycol (PEG) and QIAprep Spin Miniprep Kit were used to prepare the plasmid pGLB-G6 carrying the 5'flanking region of NGAL gene to study the nonspecific degradation of Exonuclease(ExoⅢ), the best method of preparing the plasmid ,not susceptible to ExoⅢ, would be screened. ExoⅢ deletion reaction was generated at 22 ℃ and 37 ℃ respectively and the agarose gel was run to determine the extent of digestion. PCR amplification and restriction endonuclease digestion was used to identify deleted DNA fragments. RESULTS: The plasmid extracted by QIAprep Spin Miniprep Kit is insusceptible to ExoⅢ and suitable for deletion reaction. The agarose gel electrophoresis showed that DNA fragments had not obviously shortened at 22 ℃, but at 37 ℃. Restriction endonuclease digestion is needed for further the identification of deleted DNA fragments. CONCLUSION: The quality of plasmid DNA is a crucial factor to generate specific deletion reactions of Exo III, the plamid extracted by QIAprep Spin Miniprep Kit is satisfactory; setting a control of 37 ℃ may ensure deletion reactions have happened and by PCR amplification and restriction endonuclease digestion, real deleted DNA can be attained.

Key words: nested deletion, influencing factors, NGAL gene