dgge (变形梯度凝胶电泳)研究方法及研究现状.ppt

DGGE (变性凝胶梯度电泳）,the methods of detecting single base mutations 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 (TTGE,时间温度梯度电泳).,1. DGGE 2. the principle of DGGE 3. the process of DGGE 4. the article about DGGE,1. Denaturing Gradient Gel Electrophoresis (DGGE) DGGE is an electrophoretic method to identify single base changes in a segment of DNA. Separation techniques on which DGGE is based were first described by Fischer and Lerman. DGGE is a tool for qualitatively assessing genotypic diversity in microbialecology studies (Muyzer and Smalla 1998).,In a denaturing gradient acrylamide gel, double-stranded DNA is subjected to an increasing denaturant environment and will melt in discrete segments called “melting domains“.,2.the principle of DGGE,The melting temperature (Tm) of these domains is sequence-specific. 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.,In DGGE, the denaturing environment is created by a combination of uniform temperature, typically between 50 and 65 °C and a linear denaturant gradient formed with urea and formamide. A solution of 100% chemical denaturant consists of 7 M urea and 40% formamide.,Since the Tm of a particular melting domain is sequence-specific, the presence of a mutation will alter the melting profile of that DNA when compared to wild-type. DNA containing mutations will encounter mobility shifts at different positions in the gel than the wild-type. If the fragment completely denatures, then migration again becomes a function of size(Fig 1),Fig.1. An example of DNA melting properties in a perpendicular denaturing gradient gel. At a low concentration of denaturant, the DNA fragment remains double-stranded, but as the concentration of denaturant increases, the DNA fragment begins to melt. Then, at very high concentrations of denaturant, the DNA fragment can completely melt, creating two single strands.,The denaturing gradient may be formed perpendicular or parallel to the direction of electrophoresis. （1）A perpendicular gradient gel, in which the gradient is perpendicular to the electric field, typically uses a broad denaturing gradient range, such as 0100% or 2070%. （2）In parallel DGGE, the denaturing gradient is parallel to the electric field, and the range of denaturant is narrowed to allow better separation of fragments. Examples of perpendicular and parallel denaturing gradient gels with homoduplex and heteroduplex fragments are shownin Figure 2,Fig. 2. A. Perpendicular denaturing gradient gel B. Parallel denaturing gradient gel,Collect sample containing microbial DNA to be analysed PCR amplifies a variable sequence of DNA using rRNA primers (common across species) GC clamp is attached DNA sequences loaded into wells of the DGGE gel DGGE: an electrical current causes DNA migration through the gel which contains an increasing concentration of denaturant DNA from different microbes varies in its chemical properties and stability, so the strands will be separated at different concentrations of denaturant Denatured strands stop moving through the gel The number of bands displayed indicates the number of microbes present in the sample,3. the process of DGGE,Sample Preparation It is important to optimize the PCR reaction to minimize unwanted products which may interfere with gel analysis. PCR products should be evaluated for purity by agarose gel electrophoresis before being loaded onto a denaturing acrylamide gel. perpendicular denaturing gel, load about 13 µg of amplified DNA per well(usually 50% of a 100 µl PCR volume from a 100 ng DNA template). parallel denaturing gel, load 180300 ng of amplified DNA per well (usually 510%of a 100 µl PCR volume from a 100 ng DNA template).,The addition of a 3040 base pair GC clamp to one of the PCR primers insures that the region screened is in the lower melting domain and that the DNA will remain partially double-stranded.,The size of the DNA fragments run on a denaturing gel can be as large as 1 kb in length, but only the lower melting domains will be available for mutation analysis. For complete analysis of fragments over 1 kb in length, more than one PCR reaction should be performed.,The mutant and wild-type fragments are typically amplified by the polymerase chain reaction (PCR) to make enough DNA to load on the gel. Reagent Preparation. The concentration of denaturant to use varies for the sample. The concentration of acrylamide can also vary, depending on the size of the fragment analyzed. Reagents for casting and running a DGGE gel are included .,Running DGGE,Op