变性梯度凝胶电泳(PCR-DGGE)

变性梯度凝胶电泳（DGGE）: 一种分离相似大小DNA片段 的电泳方法。即双链DNA在变性剂(如尿素和甲酰胺)浓度或 温度梯度增高的凝胶中电泳，随变性剂浓度升高，由于Tm 值不同，DNA的某些区域解链，降低其电泳泳动性，导致 迁移率下降，从而达到分离不同片段的目的。
由于各类微生物（如细菌和古细菌）的16sRNA基因序 列中可变区的碱基顺序有很大的差异，其中不同土壤微生 物的16sRNA基因的V3区扩增的DNA片断在DGGE中的应用最为 广泛，根据电泳条带的多寡和条带的位置可以初步辨别出 样品中微生物的种类多少，粗略分析土壤样品中微生物的 多样性。


In a denaturing gradient acrylamide gel, doublestranded DNA is subjected to an increasing denaturant environment and will melt in discrete segments called "melting domains". The melting temperature (Tm) of these domains is sequencespecific. When the Tm of the lowest melting domain is reached, the DNA will become partially melted, creating branched molecules. Partial melting of the DNA reduces its mobility in a polyacrylamide gel.
The methods of detecting single base mutat ions
1. Southern blotting 2. Single-Strand Conformational Polymorphism (SSCP,单链 构象多态性分析) 3. Denaturing Gradient Gel Electrophoresis (DGGE,变性凝胶 梯度电泳) 4. Carbodiimide(CDI，碳化二亚胺检测法) 5. Chemical Cleavage of Mismatch (CCM，化学切割错配法) 6. Rnase cleavage（RNA酶裂解法） 7. Heteroduplex analysis（异源双链分析） 8. the ProteinTruncation Test(PTT，蛋白截短测试) 9. Temporal Temperature Gradient Gel Electrophoresis (TTG E,时间温度梯度电泳).
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1. Microbiological characterisation of Robiola di Roccaverano cheese using PCR-DGGE. Bonetta, S.Carraro, E.Rantsiou, K. Cocolin, L. 2008 Food Microbiology.(IF 2.847) 2. Variation in the active diazotrophic community in rice paddy - nifH PCR-DGGE analysis of rhizosphere and bulk soil. Wartiainen, I. ;Eriksson, T. ;Zheng, W. W. ;Rasmussen, U. 2008 Applied Soil Ecology.(IF2.247) 3. Analysis of community structure of a microbial consortium capable of degrading benzo(a)pyrene by DGGE. Luo, Y. R. ; Tian, Y. ;Huang, X. ;Yan, C. L. ;Hong, H. S. ;Lin, G. H. ; Zheng, T. L. 2009 Marine Pollution Bulletin(IF 2.63)
2011年9月17日
变性梯度凝胶电泳(denatured gradient gel electrophoresis，DGGE)最初是Fisher和Lerman 等人 于20 世纪80 年代初期发明的，起初主要用来检测 DNA 片段中的点突变。Muyzer 等人在1993 年首次 将其应用于微生物群落结构研究 。后来又发展出其 衍生技术，温度梯度凝胶电泳（temperature gradient gel electrophoresis，TGGE）。此后十年间，该技术 被广泛用于微生物分子生态学研究的各个领域，目 前已经发展成为研究微生物群落结构的主要分子生 物学方法之一。
7. Characterization and biotechnological potential of petrol eum-degrading bacteria isolated from oil-contaminated soi ls. Zhang, Z. Z. ;Gai, L. X. ;Hou, Z. W. ;Yang, C. Y. ;Ma, C. Q. ;Wang, Z. G. ;Sun, B. P. ;He, X. F. ;Tang, H. Z. ;Xu, P. Bioresource Technology(IF 4.253)
Theቤተ መጻሕፍቲ ባይዱarticle about DGGE
① the papers which titles contain ‘DGGE’ in 2001-2010
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Since the Tm of a particular melting domain is sequ ence-specific, the presence of a mutation will alter the melting profile of that DNA when compared to wild-ty pe. DNA containing mutations will encounter mobility shifts at different positions in the gel than the wild-typ e. If the fragment completely denatures, then migratio n again becomes a function of size(Fig 1) In DGGE, the denaturing environment is created by a combination of uniform temperature, typically bet ween 50 and 65℃ and a linear denaturant gradient f ormed with urea and formamide. A solution of 100% chemical denaturant consists of 7 M urea and 40% fo rmamide.
Fig.1. An example of DNA melting properties in a perpendicular denaturing gradient gel. At a low concentration of denaturant, th e DNA fragment remains double-stranded, but as the concentrati on of denaturant increases, the DNA fragment begins to melt. Th en, at very high concentrations of denaturant,the DNA fragment c an completely melt, creating two single strands.
6. Comparative analyses of amplicon migration behavior in differing denaturing gradient gel electrophoresis (DGGE) systems. Thornhill, D. J. ;Kemp, D. W. ;Sampayo, E. M. ; S chmidt, G. W. 2010 Coral Reefs(IF 3.056)
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② the papers which themes contain ‘DGGE’ in 2001-2010
year Percent% number 2001 4.63% 184 2002 4.48% 178 2003 6.06% 241 2004 7.52% 299 2005 9.23% 367 2006 10.99% 437 2007 13.13% 522 2008 15.39% 612 2009 18.86% 750 2010 9.73% 387 in all 100% 3977
The denaturing gradient may be formed perpendicula r or parallel to the direction of electrophoresis. （1）A perpendicular gradient gel, in which the gradi ent is perpendicular to the electric field, typically uses a broad denaturing gradient range, such as 0–100% or 20–70%. （2）In parallel DGGE, the denaturing gradient is par allel to the electric field, and the range of denaturant i s narrowed to allow better separation of fragments. Examples of perpendicular and parallel denaturing gr adient gels with homoduplex and heteroduplex fragm ents are shownin Figure 2
4. Bulk soil and rhizosphere bacterial community PCRDGGE profiles and beta-galactosidase activity as ind icators of biological quality in soils contaminated by heavy metals and cultivated with Silene vulgaris (Moe nch) Garcke. Martinez-Inigo, M.J. ; PerezSanz, A. ; Ortiz, I. ;Alonso, J. ;Alarcon, R. ;Garcia, P. ;Lobo, M. C. Chemosphere(IF3.253) 2009 5. Application of real-time PCR, DGGE fingerprinting, a nd culture-based method to evaluate the effectivene ss of intrinsic bioremediation on the control of petrol eum-hydrocarbon plume. Kao, C. M. ;Chen, C. S. ;T sa, F. Y. ;Yang, K. H. ;Chien, C. C. ;Liang, S. H. ;Ya ng, C. A. ;Chen, S. C. 2010 Journal of Hazardous Materials(IF 4.144)