利用荧光标记引物和DNA自动测序仪确定DNA的断裂位点

建立了利用荧光标记引物和DNA自动测序仪进行DNA断裂位点分析的新方法, 该方法简便易行、灵敏度高、重复性好、数据分析客观性强、结果可靠, 适用于各种因素造成的DNA断裂位点的分析.

Determineation of DNA Cleavage Site Sequence Using Fluorescence Labeled Primer and DNA Sequencer

Determining the base sequence of DNA broken site is quite crucial for the study on the cleavage site specificity and mechanism of various natural or synthetic DNA cleavage regents,and on developing novel therapeutic drugs targeting at DNA.The most frequently used method depending on chemical reactions of the Maxam-Gilbert procedure,and the late arising methods used by Rui Ren et al.which were based on Sanger's DNA sequencing strategy,all had some deficiencies,either the pollution of radioactive materials,or really complicated and difficult to operate.In the present paper,a new method for DNA cleavage site sequence determination was developed.The fluorescence FAM-labeled primer was annealed to the DNA fragments,which has been cleaved by restriction enzymes or other regents,and extended along the template sequence.The products then loaded onto the polyacrylamide electrophoresis gel of ABI 377 DNA Sequencer.Data was collected and analyzed by using ABI PRISM Data Collection Software and ABI PRISM Sequencing Analysis Software.It is proved to be a credible and simple new approach to determine the base sequence of DNA broken sites.