Literature Review for Denaturing Gradient Gel Electrophoresis

Denaturing Gradient Gel Electrophoresis (DGGE) is a robust process by which point mutation can be detected. It depends upon polymerase chain reaction (PCR) products which denature at different temperatures depending upon if they contain homoduplex or different products from both wild type and mutated genes or heteroduplex or combined strains of wild type and mutated DNA strains. Heteroduplex products contain a mismatch and therefore melt more quickly than heteroduplex products (Roelfsema and Peters, 2005, pp. 79).DGGE is used most effectively to identify point mutations in genomic DNA that cause genetic diseases, to identifiy previously unidentified mutations in recessive genes, analysis of DNA from cancer tumors, and by targeting RNA, assessing the number and type of bacteria species in soil, water, and the human body (ibid, pp. 84). The DGGE process has many complicated steps that entail trial and error starting with designing the proper Gradient Gel so that the PCR product will en ter one end as a double strand, quickly denature and then stop progressing and stick in place.First the PCR product must be designed using special software that analyses the melting curves of possible PCR products. In order to keep the DNA stuck in place once the denaturing has occurred, a GC clamp consisting of a string of 40-60 nucleotides must be attached to the PCR primer resulting in a high denaturing temperature at one end and not the other. In addition, the Gradient gel must be prepared properly with a 30% gradient and adjusted so that the DNA gets trapped directly in the middle (ibid, 80).Finally, in the most technically difficult step, a constant temperature of 60 degrees Celsius must be achieved in which to perform the electrophoresis. After this the gels are soaked in a 0. 5XTAE containing ethidium bromide to visualize the DNA. Once these steps are completed successfully, the results are very clear, and seen quickly. If large numbers of samples have to be screened, DGGE i s very reliable and cost effective(ibid, 85). The following is a summary of some of the recent uses of DGGE and findings of researchers who have been exploring new territory in their fields through the use of DGGE.This review aims to highlight the strengths and weaknesses of DGGE and its most effective applications. In a study led by J. Walter of the University of Stuttegart in Germany, 16S rhibosomal primers were also used to detect lactic acid bacteria in human faeces. Subjects were given the probiotic strain lactobacillus rhamnosus DR20 to drink, and while cultural methods only detected the strain in one of the subjects, DGGE detected it in both.In addition, the food associated samples did not appear in the rosa agar cultures, but were apparent in the DGGE profile (Walter, J et al, 2001). This shows the sensitivity and reliability of DGGE methods, and its applicability to human microbiology. Another study of faeces conducted by Maukonen et al. concluded that DGGE was an effective way to establish the stability of certain groups of gastrointestinal bacteria. They successfully established the stability and diversity of the Erecta group by using DGGE to study the bacteria from 12 subjects (Maukonen et al, 2002).Applications to the study of animal faeces uncovered that DGGE is effective in identifying complex systems such as Heliobacters which are difficult to culture (Al-Soud et al, 2003). At the University of Wales in Cardiff, a team of researchers led by Charlotte E. Davies compared bacterial microfloras of healing and nonhealing chronic venous leg ulcers using both cultural and 16S rhibosomal PCR-DGGE methods. PCR-DGGE analysis found a much higher load of pseudonomads in nonhealing wounds than was apparent in cultural analysis alone (Davies, C et al, 2003).This proves the applicability of DGGE to human microbiology and its usefulness in identifying causes of illness. Similar results were obtained in studying Hypophatasia at the Barnes-Jewish Hospital Resear ch Institute. DGGE was used to identify mutations in severely affected patients and was found 100% effective in identifying recessive mutations. In addition it identified eight new mutations and one new polymorphism of hypophatasia confirming its genotypic variability (Mumm, S et al, 2002).DGGE can therefore be a powerful tool in diagnosing hypophatasia and other genetic diseases. Italian biologists used 16S rDNA PCR-DGGE and ISR-PCR methods as tools to differentiate between strains of staphyloccus isolated from fermented sausages. They obtained species specific profiles using DGGE and combining the two methods allowed them to identify 10 species and an additional 7 groups. They concluded that combining the two microbial techniques was what led to their success (Blaiotta, G et al, sep 2003).Corroborating this evidence of the need to use DGGE in combination with other techniques when studying food microbiology, a study of Cassava root fermentation in Brazzaville, Congo determined tha t the most effective method of isolating and identifying microbial communities in Cassava starch fermentation was to combine culture and DGGE methods. They found that DGGE failed to detect pure cultures recovered from enrichment and yet detected other species not apparent in any of the cultural methods used (Miambi, E, Guyote, JP and Ampe, F. , 2003).These results suggest that DGGE, while reliable and sensitive, is dependant upon other methods to complete a profile of the microbiological communities. Strides have been made to understand and identify the ecology of microbial communities, such as the work done at the University of Nottingham. Researchers used PCR-DGGE analysis focused on the V3 and V4-V5 regions of 16S genes to identify and lactobacillus and Staphlyococcus bacteria in stilton cheese. They then used Florescence in Situ Hybridization (FISH) experiments to identify the spatial arrangement of microbial species in the dairy matrix.This allowed them to conclude that there a re specific ecological reasons for microbial growth in cheese, and that there are real applications of the combination of DGGE and FISH to optimize food fermentation and preservation of traditional products (Ercolini, Hill, Dodd, Jul 2001). DGGE also has applications to farm production as seen in de Olivera et al’s study of soil rhibosomes, which concluded that DGGE provides fingerprinting of rhibosomes useful in determining the effect of agricultural practices on soils.This can help in the proper amendment of soils and monitoring of pesticides (de Olivera et al, 2006). Recognizing the usefulness of DGGE in identifying and categorizing microbial communities, and the need for more effective identification of which DNA regions to study, Zhongtang Yu and Mark Morrison performed a test to compare DGGE profiles across hypervariable (V) regions taken from the same DNA regions, and identify the most useful V regions to study in gastrointestinal microbiomes.Their recommendation is th at amplification of the V3 or V1 regions of rrs genes gives the best result, but when doing a longer amplification, the V3 to V5 or V6 to V8 range should be targeted (Yu and Morrison, 2004). DGGE has been used successfully in oceanography to identify and isolate protists that are so small they lack taxonomic features and are too unstable to be studied by traditional means.Biologists at the Woods Hole Oceanographic Institution studied protistan assemblages from the Antarctic using DGGE and were able to determine that microenvironments significantly impact assemblages and that significant genetic diversity exists in each microenvironment (Gast, Dennett and Caron, 2004). Building on the sensitivity of DGGE in identifying genetic differences, biologists in Germany identified an entirely new phylogenic group of Eukariyotic bacteria in the deeper layers of tidal flats. Their technique included using primers targeted at the 18S rRNA gene.They were also able to establish distant relationshi ps between Eukaria and grazers and deposit feeders, proving DGGE’s applicability to taxonomy. Scientists at the University of Montana also recognized DGGE’s usefulness in identifying unculturable communities and developed a way to make these communities more visible by DGGE. They first put the communities through GC fractionalization to make the study size smaller which allowed previously undetectable or underrepresented bands from the full community analysis to be seen (Holbien et al, 2004).Seeking to improve the sensitivity and versatility of DGGE’s application to microbial ecology as well as provide a way to compare and standardize gradient gels, Neufeld and Mohn of the University of British Columbia tested the use of Fluorophore-Labled primers. They found that fluorophore greatly helped intralane normalization, was relatively cheap, and allows DGGE versatility including running RNA and DNA derived patterns in the same lane (Neufeld and Mohn, 2005). References Al-Soud, Waleed Abu; Bennedsen, Mads; On, Stephen L. W.; Ouis, Ibn-Sina; Vandamme, Peter; Nilsson, Hans-Olof; Ljungh, Asa; Wadstrà ¶m, Torkel Bimal D. M. Theophilus (May 2003) Assessment of PCR-DGGE for the identification of diverse Helicobacter species, and application to faecal samples from zoo animals to determine Helicobacter prevalence. PCR Mutation Detection Protocols, Methods in Molecular Biology Volume 52, p.765-771. Blaiotta G, Pennacchia C, Ercolini D, Moschetti G, Villani F. (Sep 2003) Combining denaturing gradient gel electrophoresis of 16S rDNA V3 region and 16S-23S rDNA spacer region polymorphism analyses for the identification of staphylococci from Italian fermented sausages. Syst Appl Microbiol. 26(3):423-33 Davies, Charlotte, Katja E. Hill, Katja, Wilson, Melanie, Stephens, Phil, Hill, C. Michael, Harding, Keith and Thomas, David (Aug 2004) Use of 16S Ribosomal DNA PCR and Denaturing Gradient Gel Electrophoresis for Analysis of the Microfloras of Healing and Nonhealing Chronic Venous Leg Ulcers Journal of Clinical Microbiology, Vol. 42, No. 8, p. 3549-3557 de Oliveira, Valà ©ria,   Manfio, Gilson, Heitor Luiz da Costa Coutinho, Heitor Keijzer-Wolters, Anneke and van Elsas, Jan. (Apr 2006)Ribosomal RNA gene intergenic spacer based PCR and DGGE fingerprinting method for the analysis of specific rhizobial communities in soil Applied and Environmental Microbiology, Vol 72, No. 4 p. 2756-2764 Ercolini D, Hill PJ, Dodd CE. (Jun 2003) Bacterial community structure and location in Stilton cheese.   Appl Environ Microbiol.;69(6):3540-8. Gast, Rebecca J., Dennett, Mark and Caron, David (Apr 2004) Characterization of Protistan Assemblages in the Ross Sea, Antarctica, by Denaturing Gradient Gel Electrophoresis Applied and Environmental Microbiology, Vol 70, No. 4. p. 2028-2037 Holben, Willam,   Feris, Kevin, Kettunen, Anu and Apajalahti, Juha. (Apr 2004) GC Fractionation Enhances Microbial Community Diversity Assessment and Detection of Minority Populations of Bacteria by Denaturing Gradient Gel Electrophoresis. Applied and Environmental Microbiology, Vol 70, No. 4 p. 2263-2270 Jeroen H. Roelfsema and Dorien J. M. Peters (2005), Denaturing Gradient Gel Electrophoresis (DGGE), Medical Biomethods Handbook, p.79-85 Maukonen, Johanna, Mà ¤ttà ¶, Jaana, Satokari, Reetta,   Sà ¶derlund, Hans, Mattila-Sandholm, Tiina and Saarela, Maria (2006) PCR DGGE and RT-PCR DGGE show diversity and short-term temporal stability in the Clostridium coccoides–Eubacterium rectale group in the human intestinal microbiota. FEMS Microbiology Ecology (Online early). Miambi E, Guyot JP, Ampe F. (Apr 2003) Identification, isolation and quantification of representative bacteria from fermented cassava dough using an integrated approach of culture-dependent and culture-independent methods. Int J Food Microbiol. 25;82(2):111-20. Mumm S, Jones J, Finnegan P, Henthorn PS, Podgornik MN, Whyte MP.   (Feb. 2002) Denaturing gradient gel electrophoresis analysis of the tissue nonspecific alkaline phosphatase isoenzyme gene in hypophosphatasia. Mol Genet Metab. 75(2):143-53. Neufeld, Josh and Mohn, William. (Aug 2005) Fluorophore-Labeled Primers Improve the Sensitivity, Versatility, and Normalization of Denaturing Gradient Gel Electrophoresis Applied and Environmental Microbiology, Vol.71, No. 8 p. 4893-4896 Walter J, Hertel C, Tannock GW, Lis CM, Munro K, Hammes WP. (Jun 2001) Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using group-specific PCR primers and denaturing gradient gel electrophoresis. Appl Environ Microbiol. 2001 Jun;67(6):2578-85 Yu, Zhongtang and Morrison, Mark. (Aug 2004)Comparisons of Different Hypervariable Regions of rrs Genes for Use in Fingerprinting of Microbial Communities by PCR-Denaturing Gradient Gel Electrophoresis. Applied and Environmental Microbiology, August 2004, p. 4800-4806, Vol. 70, No. 8