Temperature Gradient Gel Electrophoresis


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Temperature Gradient Gel Electrophoresis

  1. 1. TGGETemperature Gradient Gel Electrophoresis Delivered By: Shraddha Bhatt PhD (Microbiology) AAU,Anand.
  2. 2. Points to be discussed…. Introduction Methodology Sensitivity Case studies Advantages Limitations Conclusion References 2
  3. 3. Introduction Temperature Gradient Gel Electrophoresis is a powerful technique for the separation of nucleic acids or proteins. TGGE was first developed by Lerman and Andersen of Georgia using a Beryllium Oxide plate as a thermal diffuser. (BeO has a very high thermal conductivity) TGGE method is broad and covers all disciplines which use molecular biology methods: e.g. Oncology, Virology, Immunology , RNA Viroid Research , Prion Research , Population Analysis . The TGGE method has also been used for quantitative analysis in industry and for conformational analysis of proteins. 3
  4. 4. This technique is used to: profile community complexity, to study population dynamics in microbial communities, to study differential gene expression in mixed populations, to monitor enrichment cultures, to compare DNA extraction methods, to screen clone libraries for redundancy and to determine rRNAoperonmicroheterogeneity Muyzer et al., (1998) 4
  5. 5. Study of community Dynamics 5 Study of Niche Differentiation TGGE is "sequence dependent, size independent method"
  6. 6. The first is how the structure of DNA changes with temperature; the second is how these changes in structure affect the movement of DNA through a gel DNA is a negatively charged molecule (anion) and in the presence of an electric field, will move to the positive electrode A gel is a molecular mesh, with holes roughly the same size as the diameter of the DNA string. In the presence of the electric field, the DNA will attempt to move through the mesh, and for a given set of conditions, the speed of movement is roughly proportional to the length of the DNA molecule — this is the basis for size dependent separation in standard electrophoresis. As one raises the temperature, the two strands of the DNA start to come apart; this is melting. At some high temperature, the two strands will completely separate. However, at some intermediate temperature, the two strands will be partly separated with part of the molecule still double stranded and part single stranded mobility of the DNA molecule through the gel decreases drastically when these partially melted structures are formed A very simple, but realistic analogy is to consider a person moving through a crowded room; when you extend your arms out, your movement through the room slows drastically, even though your mass has not changed. 6
  7. 7. Method of TGGE Culture independent molecular method 7
  8. 8. Casting the Gels - This step requires the individual to prepare the cuvettes for the machine, prepare the gel for the machine and pour the gel for electrophoresis. Because a buffered system must be chosen, it is important that the system remain stable within the context of increasing temperature. Thus, urea is typically utilized for gel preparation. The amount of urea & formamide(denaturants) used will have an impact on the overall temperature required to separate the DNA (Biometra, 2000). Depending on which type of TGGE is to be run, either perpendicular or parallel, varying amounts of sample need to be prepared and loaded. A larger amount of one sample is used with perpendicular, while a smaller amount of many samples are used with parallel TGGE. Electrophoresis – The gel is loaded, the sample is placed on the gel according to the type of gel that is being run—i.e. parallel or perpendicular—the voltage is adjusted and the sample can be left to run (Biometra, 2000). Staining – Once the gel has been run, to keep the results stable and further to be able to read them, the gel must be stained. While there are a number of stains that can be used for this purpose, silver staining has proven to be the most effective tool (Biometra, 2000). Elution of DNA – In this step the DNA can be eluted from the silver stain for further analysis through PCR amplification (Biometra, 2000). 8
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  10. 10. Methodology 1) For gel pouring a simple sandwich of two glass plates with a gel support film is clamped together. No need for spacers and combs For gel pouring a simple sandwich of two 2) Pouring the polyacrylamide solution takes only 1 minute. After 1.5 h the polymerized gel fixed to the support film can be retrieved from the sandwich. 3) The gel is placed on top of the metal gradient plate. A few drops of a thermal coupling solution are spread on the metal plate to optimize the thermal contact to the gel. 10
  11. 11. 11 4) Horizontal polyacrylamide gel electrophoresis of up to 18 samples takes only 30 min – 1 h, sometimes less. Pre-cut electrode wicks soaked into running buffer allow a semi-dry mode of electrophoresis. 5) The polyacrylamide gel is stained by a rapid silver staining protocol. Perpendicular TGGE Parallel TGGE
  12. 12. Sensitivity: Small amount of material used for separation, DNA or RNA fragments appear as fine bands which can be clearly distinguished from each other. Complex band pattern can be ananlyzed due to the high resolution capability of the gradient block. TGGE detects mutations in mixed DNA samples. Whenever Heterozygous DNA is to be analysed, direct sequencing will not give a clear signal at the position of the mutation. This is especially the case if the mutated gene is masked by a high background of normal cells. TGGE reliably detects mutations in a 1:10 dilution(and higher) of wild type DNA. 12
  13. 13. Case studies “Hunting for Microbes” with Molecular Tools 13
  14. 14. Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology TGGE analysis of PCR-amplified 16SrDNA fragments has been applied to compare bacterial populations inhabiting the rhizosphere and phyllosphere of transgenic potato plants expressing T4- lysozyme and non-transgenic potato plants(in figure). TGGE patterns of rDNA fragments from the rhizosphere were complex, but identical between transgenic and non- transgenic plants. In contrast, profiles obtained from the phyllosphere samples were less complex, but showed much more variation between plants. Muyzeret al., (1998) 14
  15. 15. TGGE was used to describe the diversity of methanogenmcrA sequences and the differences in community structure between samples. Results indicate that sludge application may reduce soil methanogen community diversity. Sheppard et al., (2005) 15
  16. 16. Identification of Lactobacillus spp. isolated from naturally fermented Italian sausages using temperature gradient gel electrophoresis. Thirty-nine strains of Lactobacillus spp. were isolated from naturally fermented sausages and, after traditional identification, were tested by the PCR–TGGE protocol developed. Two species genetically closely related, not easily differentiated using traditional methods, but clearly identified by using the method involved Cocolinet al., (2000) 16
  17. 17. HENRI-DUBERNET et al.,(2004) Reference strains Lane I: Lactobacillus delbrueckii subsp. bulgaricus CNRZ 225; Lane II: Lactobacillus paracasei subsp. paracasei CNRZ 763; Lane III: Lactobacillus plantarum CNRZ 211 Lactobacilli community biodiversity and evolution during the production of Camembert in three cheese-making factories. Temperature gradient gel electrophoresis (TGGE) to analyse total microbial DNA and DNA from single isolates. TGGE patterns of total microbial DNA from milk and cheese showed that Lactobacillus paracasei subsp. paracasei was a dominant species in the three factories and that Lb. plantarum was also a dominant species in one. TGGE profiles from individual isolates confirmed that these two species were dominant. 17
  18. 18. C Samples of dry and slurry yeasts used in three different micro-breweries (named A, B and C) in the Friuli Venezia Giulia region (Italy) were analyzed to evaluate the contamination due to wild yeasts and lactic acid bacteria during the brewing process. Manzanoet al., (2005) 18
  19. 19. The samples used in this study were collected from the Guadarrama River, located in central Spain, near the city of Madrid. Sampling sites were selected to include locations above and below populated areas. Sampling site 1 is located in an unaffected by human influence area, upstream of an industrial and populated zone, site 2 and site 3 , 22 km and 38 km downstream, which receive industrial and domestic sewage from the nearby human settlements. The TGGE analysis showed noticeable differences in the banding patterns at the various sampling locations The number of bands was similar among replicates (different stones) from each location but clearly differed among the sites with distinct levels of pollution, whereby more bands were present in upstream than in downstream sites. The TGGE results revealed that the structure of the cyanobacterial community differed along the pollution gradient of the river. Microscopic and molecular approaches showed that cyanobacterial diversity decreased in a downstream direction. Rodrı´guez et al., (2007) 19
  20. 20. Advantages of TGGE Large number of samples can analysed simultaneously Reliable, Reproducible, Rapid, Inexpensive, Potentially find uncultivable microbes. (Simpson et al., 2000) This procedure allows direct identification of the presence and relative abundance of different species and provides a semi-quantitative estimation of the genetic diversity of microbial populations. (Vaughan et al., 1999). TGGE as a technique for studying microbial diversity is better than cloning and subsequent sequencing of PCR amplifiedrDNA. (Muyzeret al., 1993). TGGE allows the simultaneous analysis of multiple samples making it possible to follow community changes over time and space . (Muyzer 1999; Nicolaisen and Ramsing 2002). 20
  21. 21. 21
  22. 22. Limitations of TGGE 22
  23. 23. separation of only relatively small fragments, up to 500 basepairs (Myers et al.,1985).This limits the amount of sequence information for phylogenetic inferences as well as for probe design.( but can solved by hybridization as well as by group specific PCR) it is not always possible to separate DNA fragments which have a certain amount of sequence variation. Vallaeys et al.,(1997) found that 16S rDNA fragments obtained from different methane oxidizing bacteria could not be resolved by TGGE although they had substantial sequence variation. using DNA- DNA reannealing experiments Torsviket al., (1990) found that there might be as many as 104different genomes present in soil samples. It will be obvious that TGGE cannot separate all of the 16S rDNA fragments obtained from such a variety of microorganisms. Comigration of DNA fragments can be a problem for retrieving clean sequences from individual bands. study of community diversity on the basis of 16S rRNA genes, using TGGE is the presence in some bacteria of multiple rrNoperonswith sequence microheterogeneity TGGE can visualise this sequence heterogeneity which might lead to an overestimation of the number of bacteria within natural communities. 23
  24. 24. Conclusion The TGGE System is a powerful and rapid tool for distinguishing DNA fragments of the same length but different sequences from each other. It was possible to separate a complex PCR mixture in less than 3 h including a 1 h silver staining procedure The broad temperature range of the perpendicular TGGE defines the best conditions for separation in one experimental run. Subsequently, it is possible to run multiple samples in parallel TGGE at a narrower temperature range. 24
  25. 25. “However, all modern molecular techniques based on 16S/18S rRNA/rDNA cannot preclude classic microbiological techniques. It should be used together to ensure accurate results.” 25
  26. 26. Thank You… 26
  27. 27. Muyzer G. Smalla K(1998).Application of denaturing gradient gel electrophoresis(DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie van Leeuwenhoek, 73: 127-141. Sheppard KS., McCarthy JA, Loughmane PJ, Gray D.N, Head MI and Lloyd D. (2005) The impact of sludge amendment on methanogen community structure in an upland soil.Applied Soil Ecology., 28 : 147-162. Cocolin L, Manzano M, Cantoni C and Comi G. Development of a rapid method for the identification of Lactobacillus spp. isolated from naturally fermented Italian sausages using a polymerase chain reaction– temperature gradient gel electrophoresis Letters in Applied Microbiology (2000), 30: 126–129. Herna¨n-Go¨mez a S, Espinosa a J.C., Ubeda b J.F. Characterization of wine yeast by temperature gradient gel electrophoresis (TGGE).FEMS Microbiology Letters(2000) 193 : 45-50. v. Manzano M Giusto C, Bartolomeoli I, Buiatti S. and Comi B. Microbiological Analyses of Dry and Slurry Yeasts for Brewing. J. Inst. Brew. (2005). 111(2):203–208. vi. Muyzer, G., DGGE/TGGE a method for identifying genes from natural ecosystems. Curr. Opin. Microbiol.(1999).2: 317–322. vii. Simpson J.M., McCracken V.J., Gaskins H.R., Mackie R.I. Denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA amplicons to monitor changes in fecal bacterial populations of weaning pigs after introduction of Lactobacillus reuteri strain MM53. Appl. Environ. Microbiol. (2000). 66: 4705–4714. 27 References