Denatured gradient gel electrophoresis strip direct sequencing system optimization method
A denaturing gradient gel and system optimization technology, applied in biochemical equipment and methods, measuring devices, microbiological determination/inspection, etc., can solve the problems of reducing sequencing work, tediousness, and high cost, and achieve the effect of reducing sequencing costs
Inactive Publication Date: 2012-04-25
XIAMEN UNIV +1
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[0002] If the PCR products of the DGGE strip leachate are directly sent for sequencing, generally 80-90% of them will have double peaks. Therefore, in the past, the sequencing of the PCR products of the strip leachate was transformed into E.coli Topl0 competent cells by ligation, and then selected The clones were sent for sequencing ([1] Yang Guimei, Tang Wenqiao, Li Huirong et al. Analysis of Vibrio colony composition in Puffer puffer obscura by PCR-DGGE method. Journal of Shanghai Fisheries University. 2006, 15(3): 257-263; [2] Zhi-Yong Li, Li-Ming He, Jie Wu, et al. Bacterial community diversity associated with four marine sponges from the south China Sea based on 16S rDNA-DGGE fingerprinting. Journal of Experimental Marine Biology and Ecology. 2006, (329): 75-85), this method has the following disadvantages: first, the cost is high, and costs such as connecting transformation kits and competent states are required; second, it is relatively cumbersome; third, it is possible to detect other sequences
However, there are no reports to use a variety of specific optimization methods to purify bands and reduce the sequencing work of a large number of bands with the same migration distance
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[0029] Generally, many bands appear in the DGGE profile of environmental-like PCR products after recovery and purification, and they are very close to each other, such as figure 1 As shown, the strip leachate PCR products generally cannot be directly sent for sequencing after recovery and purification.
[0030] Again, DGGE can separate the impurity bands in the PCR product by changing the concentration gradient of the denaturant, such as figure 2 As shown, the bands marked with punch holes are the target bands, and the other unmarked bands are miscellaneous bands separated after DGGE again. If DGGE is not performed again and sent directly for sequencing, many results must be double peaks.
[0031]Therefore, DGGE was performed on the PCR product of the leach solution with DGGE bands again, so that the miscellaneous bands in the product were further separated, so as to obtain a single band, ensuring that the PCR recovery product of the leach solution could be sequenced. At the...
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The invention provides a system optimization method for the direct sequencing of denaturing gradient gel electrophoresis bands, and relates to a gel electrophoresis band. The invention provides a system optimization method for the direct sequencing of DGGE bands. The method comprises the steps of establishing a control map, performing DGGE twice, mixing and loading a plurality of samples, reducing the concentration gradient of denaturant, determining identical bands, using a marking sample, staggering adjacent bands and performing DGGE again. The method has the advantages of purifying the bangs, improving the sequencing effect of PCR recovery products of the bands and reducing the cost for sequencing the identical bands.
Description
technical field [0001] The invention relates to a gel electrophoresis band, in particular to a system optimization method for direct sequencing of PCR products of multiple denaturing gradient gel electrophoresis (DGGE) band leaching solutions. Background technique [0002] If the PCR products of the DGGE strip leachate are directly sent for sequencing, generally 80-90% of them will have double peaks. Therefore, in the past, the sequencing of the PCR products of the strip leachate was transformed into E.coli Topl0 competent cells by ligation, and then selected The clones were sent for sequencing ([1] Yang Guimei, Tang Wenqiao, Li Huirong et al. Analysis of Vibrio colony composition in Puffer puffer obscura by PCR-DGGE method. Journal of Shanghai Fisheries University. 2006, 15(3): 257-263; [2] Zhi-Yong Li, Li-Ming He, Jie Wu, et al. Bacterial community diversity associated with four marine sponges from the south China Sea based on 16S rDNA-DGGE fingerprinting. Journal of Exper...
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IPC IPC(8): G01N27/447C12Q1/68
Inventor 汤坤贤焦念志
Owner XIAMEN UNIV