TGGE is a technique used to separate DNA or proteins based on temperature gradients. It allows complex samples to be separated into distinct bands based on small sequence variations. The document discusses the methodology of TGGE, including gel casting and electrophoresis. Several case studies are presented where TGGE was used to analyze microbial communities in various environmental and food samples. The advantages of TGGE are its ability to analyze many samples simultaneously, detect unculturable microbes, and provide semi-quantitative data. Limitations include inability to separate all DNA fragments and potential for co-migration of bands. In conclusion, TGGE is a powerful tool for microbial community analysis when used along with traditional techniques.
Gel electrophoresis native, denaturing&reducingLovnish Thakur
Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation.
Gel electrophoresis native, denaturing&reducingLovnish Thakur
Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation.
In shotgun sequencing the genome is broken randomly into short fragments (1 to 2 kbp long) suitable for sequencing. The fragments are ligated into a suitable vector and then partially sequenced. Around 400–500 bp of sequence can be generated from each fragment in a single sequencing run. In some cases, both ends of a fragment are sequenced. Computerized searching for overlaps between individual sequences then assembles the complete sequence.
Open reading frame is part of reading frame that contains no stop codons or region of amino acids coding triple codons.
ORF starts with start codon and ends at stop codon.
RAPD markers are decamer DNA fragments.
RAPD is a type of PCR reaction.
as the name suggest it is a fast method when compared to the traditional PCR medthod.
It is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels used as support media.
Gels are made by free radical-induced polymerization of acrylamide and N,N’-Methylenebisacrylamide.
It is the most widely used technique of electrophoresis.
Automated sequencing of genomes require automated gene assignment
Includes detection of open reading frames (ORFs)
Identification of the introns and exons
Gene prediction a very difficult problem in pattern recognition
Coding regions generally do not have conserved sequences
Much progress made with prokaryotic gene prediction
Eukaryotic genes more difficult to predict correctly
This is technique used widely for protein separation from a mixture and is very easy and less costly method. Slides cover all essential points about EMSA and it is quite interesting to know that how it detect and separate different proteins and their mobility shift assay.
In shotgun sequencing the genome is broken randomly into short fragments (1 to 2 kbp long) suitable for sequencing. The fragments are ligated into a suitable vector and then partially sequenced. Around 400–500 bp of sequence can be generated from each fragment in a single sequencing run. In some cases, both ends of a fragment are sequenced. Computerized searching for overlaps between individual sequences then assembles the complete sequence.
Open reading frame is part of reading frame that contains no stop codons or region of amino acids coding triple codons.
ORF starts with start codon and ends at stop codon.
RAPD markers are decamer DNA fragments.
RAPD is a type of PCR reaction.
as the name suggest it is a fast method when compared to the traditional PCR medthod.
It is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels used as support media.
Gels are made by free radical-induced polymerization of acrylamide and N,N’-Methylenebisacrylamide.
It is the most widely used technique of electrophoresis.
Automated sequencing of genomes require automated gene assignment
Includes detection of open reading frames (ORFs)
Identification of the introns and exons
Gene prediction a very difficult problem in pattern recognition
Coding regions generally do not have conserved sequences
Much progress made with prokaryotic gene prediction
Eukaryotic genes more difficult to predict correctly
This is technique used widely for protein separation from a mixture and is very easy and less costly method. Slides cover all essential points about EMSA and it is quite interesting to know that how it detect and separate different proteins and their mobility shift assay.
This presentation contain the information about gel electrophoresis method , instruments & types.
Electrophoresis is a method through biological molecules are separated by applying an electric field.
Main purpose of this method is to determine the number , amount & mobility of biological component.
There are some internal & external factors that affects the process of electrophoresis.
The bio-molecules have charge on it & when we apply an electric field , the charge particles move to the opposite cathode. In this way, charge particles are separated
There are 3 types of gels that use in this process .
In this buffers are also used which provide ions that carry a current.
Advances in Molecular Cytogenetics: Potential for Crop Improvement.pptxKanshouwaModunshim
Title: Exploring Advances in Cytogenetics and Molecular Cytogenetics
Description:
Delve into the intricate world of cytogenetics and its cutting-edge counterpart, molecular cytogenetics, through this insightful presentation. Understand the profound relationship between chromosome structure, behavior, and gene function, with a particular focus on their relevance to crop improvement programs.
Key Points:
Introduction to Cytogenetics: Explore the fundamental principles of cytogenetics, its historical significance, and the recent influence of molecular tools, leading to the emergence of molecular cytogenetics.
Importance in Crop Improvement: Uncover the pivotal role of molecular cytogenetics in crop improvement programs, offering insights into the structural and functional organization of genomes within chromosomes.
Karyotyping: Gain a comprehensive understanding of karyotyping, its significance in identifying chromosomal abnormalities, and its applications in studying evolutionary relationships among different taxa.
Chromosome Identification and Sorting: Learn about the techniques involved in the identification and sorting of individual chromosomes, crucial steps in cytogenetics research for various crops.
Chromosome Banding Techniques: Explore different chromosome banding techniques, such as G-Banding and C-Banding, and understand their applications in detecting structural rearrangements.
CHIAS (Chromosome Image Analyzing System): Get insights into the CHIAS software and its role in mapping and identifying chromosomes automatically.
Flow Cytometry: Discover the applications of flow cytometry in detecting and measuring physical and chemical characteristics of cells, with a focus on its relevance in chromosome research.
In Situ Hybridization: Explore the technique of in situ hybridization, particularly the fluorescent variant, and its applications in precise localization of specific DNA segments.
Genomics and Whole Genome Sequencing: Delve into the realm of genomics and whole-genome sequencing, understanding the approaches like BAC to BAC and Whole Genome Shotgun.
Case Study: Uncover a case study involving the identification of a Wheat-Psathyrostachys huashanica ditelosomic addition line, showcasing the practical applications of the discussed techniques.
Conclusion: Summarize the key takeaways from the presentation, emphasizing the role of these techniques in advancing precision breeding and crop improvement.
Flow Cytometric Analysis for Ploidy and DNA Content of Banana Variants Induce...paperpublications3
Abstract: Nuclear DNA content of mutated banana plants was determined by using flow cytometric techniques. It is a powerful tool for large scale screening of ploidy levels. Nuclei were isolated from young leaves from (banana mutants & Glycine plants) supplemented with Propidium- iodide (PI) and RNAse. "Glycine max" used as internal reference standard for identifying the nuclear DNA content by FCM. For ploidy estimation DAPI was used. The results showed differences in DNA content between variants indicating the effect of gamma-irradiation on the genotype of these plants. Variants of short plant stature or stunted growth showed great differences in DNA content compared to control (non-irradiated). The phenotypic variations observed at high doses were likely due to changes in the DNA sequences at the chromosomal level. Nuclear DNA contents decreased with an increase of gamma-dose from 20 Gy to 60 Gy. However, there were no significant differences between DNA content at 20 Gy and 30 Gy and also between 40 Gy and 60 Gy, while they were differed significantly from the control. The results showed no significant differences in ploidy level between all samples used (3n); while all selected mutants (variants) showed differences in DNA content.
Abstract
Objective(s):
The development of reliable and ecofriendly process for the synthesis of nano-metals is an important aspect in the field of nanotechnology. Nano-metals are a special group of materials with broad area of applications.
Materials and Methods:
In this study, extracellular synthesis of silver nanoparticles (SNPs) performed by use of the gram positive soil Streptomycetes. Streptomycetes isolated from rice fields of Guilan Province, Iran (5 isolates). Initial characterization of SNPs was performed by visual change color. To determine the bacterium taxonomical identity, its colonies characterized morphologically by use of scanning electron microscope. The PCR molecular analysis of active isolate represented its identity partially. In this regard, 16S rDNA of isolate G was amplified using universal bacterial primers FD1 and RP2. The PCR products were purified and sequenced. Sequence analysis of 16S rDNA was then conducted using NCBI GenBank database using BLAST. Also SNPs were characterized by, transmission electron microscopy (TEM) and X-ray diffraction spectroscopy (XRD).
Results:
From all 5 collected Streptomyces somaliensis isolates, isolate G showed highest extracellular synthesis of SNPs via in vitro. SNPs were formed immediately by the addition of (AgNO3) solution (1 mM). UV-visible spectrophotometry for measuring surface plasmon resonance showed a single absorption peak at 450 nm, which confirmed the presence of SNPs. TEM revealed the extracellular formation of spherical silver nanoparticles in the size range of 5-35 nm.
Conclusions:
The biological approach for the synthesis of metal nanoparticles offers an environmentally benign alternative to the traditional chemical and physical synthesis methods. So, a simple, environmentally friendly and cost-effective method has been developed to synthesize AgNPs using Streptomycetes.
Targeted Induced Local Lesions IN Genome. Mutations (Single base pair substitution) are created by traditionally used chemical mutagens. Identify SNPs and / or INDELS in a gene / genes of interest from a mutagenized population.
Transcript: Selling digital books in 2024: Insights from industry leaders - T...BookNet Canada
The publishing industry has been selling digital audiobooks and ebooks for over a decade and has found its groove. What’s changed? What has stayed the same? Where do we go from here? Join a group of leading sales peers from across the industry for a conversation about the lessons learned since the popularization of digital books, best practices, digital book supply chain management, and more.
Link to video recording: https://bnctechforum.ca/sessions/selling-digital-books-in-2024-insights-from-industry-leaders/
Presented by BookNet Canada on May 28, 2024, with support from the Department of Canadian Heritage.
Neuro-symbolic is not enough, we need neuro-*semantic*Frank van Harmelen
Neuro-symbolic (NeSy) AI is on the rise. However, simply machine learning on just any symbolic structure is not sufficient to really harvest the gains of NeSy. These will only be gained when the symbolic structures have an actual semantics. I give an operational definition of semantics as “predictable inference”.
All of this illustrated with link prediction over knowledge graphs, but the argument is general.
UiPath Test Automation using UiPath Test Suite series, part 4DianaGray10
Welcome to UiPath Test Automation using UiPath Test Suite series part 4. In this session, we will cover Test Manager overview along with SAP heatmap.
The UiPath Test Manager overview with SAP heatmap webinar offers a concise yet comprehensive exploration of the role of a Test Manager within SAP environments, coupled with the utilization of heatmaps for effective testing strategies.
Participants will gain insights into the responsibilities, challenges, and best practices associated with test management in SAP projects. Additionally, the webinar delves into the significance of heatmaps as a visual aid for identifying testing priorities, areas of risk, and resource allocation within SAP landscapes. Through this session, attendees can expect to enhance their understanding of test management principles while learning practical approaches to optimize testing processes in SAP environments using heatmap visualization techniques
What will you get from this session?
1. Insights into SAP testing best practices
2. Heatmap utilization for testing
3. Optimization of testing processes
4. Demo
Topics covered:
Execution from the test manager
Orchestrator execution result
Defect reporting
SAP heatmap example with demo
Speaker:
Deepak Rai, Automation Practice Lead, Boundaryless Group and UiPath MVP
Securing your Kubernetes cluster_ a step-by-step guide to success !KatiaHIMEUR1
Today, after several years of existence, an extremely active community and an ultra-dynamic ecosystem, Kubernetes has established itself as the de facto standard in container orchestration. Thanks to a wide range of managed services, it has never been so easy to set up a ready-to-use Kubernetes cluster.
However, this ease of use means that the subject of security in Kubernetes is often left for later, or even neglected. This exposes companies to significant risks.
In this talk, I'll show you step-by-step how to secure your Kubernetes cluster for greater peace of mind and reliability.
UiPath Test Automation using UiPath Test Suite series, part 3DianaGray10
Welcome to UiPath Test Automation using UiPath Test Suite series part 3. In this session, we will cover desktop automation along with UI automation.
Topics covered:
UI automation Introduction,
UI automation Sample
Desktop automation flow
Pradeep Chinnala, Senior Consultant Automation Developer @WonderBotz and UiPath MVP
Deepak Rai, Automation Practice Lead, Boundaryless Group and UiPath MVP
Epistemic Interaction - tuning interfaces to provide information for AI supportAlan Dix
Paper presented at SYNERGY workshop at AVI 2024, Genoa, Italy. 3rd June 2024
https://alandix.com/academic/papers/synergy2024-epistemic/
As machine learning integrates deeper into human-computer interactions, the concept of epistemic interaction emerges, aiming to refine these interactions to enhance system adaptability. This approach encourages minor, intentional adjustments in user behaviour to enrich the data available for system learning. This paper introduces epistemic interaction within the context of human-system communication, illustrating how deliberate interaction design can improve system understanding and adaptation. Through concrete examples, we demonstrate the potential of epistemic interaction to significantly advance human-computer interaction by leveraging intuitive human communication strategies to inform system design and functionality, offering a novel pathway for enriching user-system engagements.
Smart TV Buyer Insights Survey 2024 by 91mobiles.pdf91mobiles
91mobiles recently conducted a Smart TV Buyer Insights Survey in which we asked over 3,000 respondents about the TV they own, aspects they look at on a new TV, and their TV buying preferences.
Software Delivery At the Speed of AI: Inflectra Invests In AI-Powered QualityInflectra
In this insightful webinar, Inflectra explores how artificial intelligence (AI) is transforming software development and testing. Discover how AI-powered tools are revolutionizing every stage of the software development lifecycle (SDLC), from design and prototyping to testing, deployment, and monitoring.
Learn about:
• The Future of Testing: How AI is shifting testing towards verification, analysis, and higher-level skills, while reducing repetitive tasks.
• Test Automation: How AI-powered test case generation, optimization, and self-healing tests are making testing more efficient and effective.
• Visual Testing: Explore the emerging capabilities of AI in visual testing and how it's set to revolutionize UI verification.
• Inflectra's AI Solutions: See demonstrations of Inflectra's cutting-edge AI tools like the ChatGPT plugin and Azure Open AI platform, designed to streamline your testing process.
Whether you're a developer, tester, or QA professional, this webinar will give you valuable insights into how AI is shaping the future of software delivery.
Generating a custom Ruby SDK for your web service or Rails API using Smithyg2nightmarescribd
Have you ever wanted a Ruby client API to communicate with your web service? Smithy is a protocol-agnostic language for defining services and SDKs. Smithy Ruby is an implementation of Smithy that generates a Ruby SDK using a Smithy model. In this talk, we will explore Smithy and Smithy Ruby to learn how to generate custom feature-rich SDKs that can communicate with any web service, such as a Rails JSON API.
JMeter webinar - integration with InfluxDB and GrafanaRTTS
Watch this recorded webinar about real-time monitoring of application performance. See how to integrate Apache JMeter, the open-source leader in performance testing, with InfluxDB, the open-source time-series database, and Grafana, the open-source analytics and visualization application.
In this webinar, we will review the benefits of leveraging InfluxDB and Grafana when executing load tests and demonstrate how these tools are used to visualize performance metrics.
Length: 30 minutes
Session Overview
-------------------------------------------
During this webinar, we will cover the following topics while demonstrating the integrations of JMeter, InfluxDB and Grafana:
- What out-of-the-box solutions are available for real-time monitoring JMeter tests?
- What are the benefits of integrating InfluxDB and Grafana into the load testing stack?
- Which features are provided by Grafana?
- Demonstration of InfluxDB and Grafana using a practice web application
To view the webinar recording, go to:
https://www.rttsweb.com/jmeter-integration-webinar
Elevating Tactical DDD Patterns Through Object CalisthenicsDorra BARTAGUIZ
After immersing yourself in the blue book and its red counterpart, attending DDD-focused conferences, and applying tactical patterns, you're left with a crucial question: How do I ensure my design is effective? Tactical patterns within Domain-Driven Design (DDD) serve as guiding principles for creating clear and manageable domain models. However, achieving success with these patterns requires additional guidance. Interestingly, we've observed that a set of constraints initially designed for training purposes remarkably aligns with effective pattern implementation, offering a more ‘mechanical’ approach. Let's explore together how Object Calisthenics can elevate the design of your tactical DDD patterns, offering concrete help for those venturing into DDD for the first time!
2. Points to be discussed…. Introduction Methodology Sensitivity Case studies Advantages Limitations Conclusion References 2
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. 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. Study of community Dynamics 5 Study of Niche Differentiation TGGE is "sequence dependent, size independent method"
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. Method of TGGE Culture independent molecular method 7
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
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 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. 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
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. 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. 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. 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. 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. 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. 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
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.
25. 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
26. “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
28. 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