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Gradient gel electrophoresis and pulse field gel electrophoresis
- 1. GRADIENT GEL ELECTROPHORESIS & PULSE FIELD GEL ELECTROPHORESIS By Kaviya priya. A (23PBT007)
- 2. GRADIENT GEL ELECTROPHORESIS ■ A Polyacrylamide gel system ■ Instead of running a slab gel of uniform pore size throughout (e.g. a 15% gel) a gradient gel is formed, where the acrylamide concentration varies uniformly from, typically, 5% at the top of the gel to 25% acrylamide at the bottom of the gel. TYPES OF GRADIENT GEL ELETROPHORESIS 1. Temperature gradient gel Electrophoresis 2. Denaturing gradient gel Electrophoresis
- 3. ■ Temperature gradient gel electrophoresis (TGGE) and denaturing gradient gel electrophoresis (DGGE) are forms of electrophoresis which use either a temperature or chemical gradient to denature the sample as it moves across an acrylamide gel. ■ TGGE and DGGE can be applied to nucleic acids such as DNA and RNA , and (less commonly) proteins. ■ TGGE relies on temperature dependent changes in structure to separate nucleic acids. DGGE separates genes of the same size based on their different denaturing ability which is determined by their base pair sequence. ■ DGGE was the original technique, and TGGE a refinement of it. PRINCIPLE
- 4. TEMPERATURE GRADIENT GELL ELECTROPHORESIS ■ At room temperature, the DNA will exist stably in a double-stranded form. ■ As the temperature is increased, the strands begin to separate (melting), and the speed at which they move through the gel decreases drastically. ■ Critically, the temperature at which melting occurs depends on the sequence GC base pairs are more stable than AT due to stacking interactions, not due to the difference in hydrogen bonds , so TGGE provides a "sequence dependent, size independent method" for separating DNA molecules. ■ TGGE separates molecules and gives additional information about melting behavior and stability
- 6. ■ Denaturing gradient gel electrophoresis (DGGE) works by applying a small sample of DNA (or RNA) to an electrophoresis gel that contains a denaturing agent. ■ Mixture of urea and formamide. ■ As a result of this melting, the DNA spreads through the gel and can be analyzed for single components, even those as small as 200-400bp. STEPS INVOLVED IN DGGE: 1. PCR products with GC clamp are applied on the top of the gel. 2. Double stranded DNA fragments are moving in the gel due to the electrophoretical power. 3. DNA molecules meet with increasing denaturing concentrations - partially melting, decreasing mobility. 4. DNA fragments are melted, practically GC clamp holds the strands together - molecules stop moving DENATURING GRADIENT GEL ELECTROPHORESIS
- 8. APPLICATION 1. Detection of microheterogenity in rRNA encoding genes. 2. Comparison of different DNA extraction protocols . 3. Screening of clone libraries . 4. Determining PCR and cloning biases. 5. molecular fingerprinting method for complex ecosystem communities
- 9. LIMITATIONS ■ The optimal fragment size is about 500 bp. ■ Labour-intensive nature. ■ Often less reproducibility in terms of band pattern. ■ Intensity detection after electrophoretic separation.
- 10. PULSE FIELD GEL ELECTROPHORESIS ■ Pulsed-field gel electrophoresis (PFGE) is a powerful molecular typing technique by which genomic DNA is isolated from the organism of interest, followed by restriction enzyme analysis. PRINCIPLE ■ The digestion products are then analyzed on an agarose gel by applying an electric field that periodically changes direction allowing for separation of the larger DNA fragments (entire genomic DNA) and approximate measurement of fragment length. ■ PFGE can separate large DNA molecules (up to 10 Mb), whereas standard DNA gel electrophoresis commonly resolves fragments up to 50 kb. PFGE ∼ takes 2–3 days, excluding sample preparation.
- 11. TYPES ■ There are various types of PFGE methods that are used for isolating and typing DNA with large pieces. ■ Field-Inversion Gel Electrophoresis (FIG) -provides acceptable resolution of over 800 Kb ■ Orthogonal-Field Alternation Gel Electrophoresis (OFAGE) -DNA molecules between 1000 kb and 2000 kb ■ Asymmetric Field Inversion Gel Electrophoresis (AFIGE) - This is a method for detecting DNA double-strand breaks (DSBs) using a reverse-asymmetrical field gel electrophoresis
- 12. ■ Clamped Homogeneous Electric Field (CHEF) - This system can separate molecules over 7000 kb in size. ■ Programmable Autonomously-Controlled Electrodes(PACE) - It utilizes 24 electrodes and The PACE system can separate DNA fragments from 100 bp to more than 6Mb ■ Rotating Gel Electrophoresis (RGE) - This method is considered easy to use and is suitable for DNA with a size range of 50 to 6000 Kb. ■ Pulsed-Homogeneous Orthogonal Field Gel Electrophoresis (PHOGE) - This system is specifically designed to separate DNA fragments larger than 1Mb, making it a powerful tool for larger DNA analysis.
- 14. APPLICATION ■ PFGE is considered as a gold standard technique in the area of epidemiological studies of pathogenic organisms. ■ In genome size estimation, PFGE is considered as an efficient method. ■ PFGE is also used for genotyping or genetic fingerprinting. ■ Yeast Artificial Chromosome (YAC) libraries are constructed by PFGE. ■ PFGE has also been used in the analysis of large DNA molecules from fungi and parasitic protozoa. ■ PFGE is also used in the study of radiation-induced DNA damage and repair.
- 15. LIMITATIONS ■ PFGE method consumes more time which might take overnight or a couple of days. ■ It cannot separate the fragments in every part of the gel at the same time. ■ One change in restriction site might form more than one band. ■ PFGE does not differentiate isolates to the same degree as whole genome sequencing (WGE).
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