logging in or signing up NANDU PCR Presentation nnandu1 Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 294 Category: Science & Tech.. License: All Rights Reserved Like it (0) Dislike it (0) Added: September 07, 2010 This Presentation is Public Favorites: 0 Presentation Description No description available. Comments Posting comment... By: dr.eshanidewan (10 month(s) ago) Dear Sir, I would like to have Quantitative RT-PCR presentation. Can you please send me the presentation to my mail ID eshanidewan@yahoo.com. I will be thankful to you. regards. eshani Saving..... Post Reply Close Saving..... 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See all Premium member Presentation Transcript POLYMERASE CHAIN REACTION(PCR)&APPLICATIONS N.Nanda GopalJr.Scientist : POLYMERASE CHAIN REACTION(PCR)&APPLICATIONS N.Nanda GopalJr.Scientist Overview : Overview Differences between In-vivo and In-vitro replication Basics of PCR: Definition, invention. Updates of PCR: Available techniques and applications. Principle of PCR Components of PCR and their significance. Reaction steps with description. Applications: Industrial Human Health Agricultural- Plant & animal Forensic Genomic Invention of PCR : Invention of PCR Invented by Kary Banks Mullis (1983), Cetus corp, USA. Used klenow fragment of DNA polymerase-I, Major disadvantage - sensitive to high temps > 900C. Replaced by Lawyer et.al. (1989) with Taq DNA Polymerase of Thermus aquaticus, a volcanic achaebacteria. Thermostable, high fidelity with proof reading function were identified, Eg: Vent polymerase- Thermococcus litoralis Pfu polymerase- Pyrococcus furiosus Kary Mullis : Kary Mullis Born in a farmer family 1944, North Carolina, USA. Worked as Pharmaceutical Chemist at Cetus Corp., Nobel prize in Chemistry 1993. Japan Prize. Thomas A. Edison Award. Also invented UV-sensitive plastic that changes color in response to light. Technique : Technique In-Vitro, exponential amplification of target DNA sequences Using Taq-Polymerase (obtained from Thermus aquaticus), Primers- To initiate and to avoid non-specific DNA amplification flanked either side of the target. By controlled cycling the temperature, the target DNA is repetitively denatured and copied. A single copy of the target DNA can be amplified to obtain billions of replicates. To amplify RNA sequences, must be converted to DNA via reverse transcriptase. This two-phase procedure is known as ‘RT-PCR’. Components of PCR & significance : Components of PCR & significance Nucleiotides (dNTPs)- Building blocks Primers set (Forward & Reverse)- Initiation DNA Polymerase enzyme- Polymerization MgCl2- Co-factor for DNA Polymerase PCR Buffer- Stabilization of the reaction mix PCR Mix : PCR Mix Basic steps of PCR : Basic steps of PCR Thermal denaturation of the ds DNA at 940C (30 sec) Annealing of primers to their complementary sequences 50-600C(30sec) Extension at 720C (5 min) of the annealed primers with DNA polymerase. (Earlier was provided in different water baths) The amplified product is called “Amplicon”. Versatile technique with high speed, efficiency, sensitivity and specificity Eliminates the laborious conventional culture methods. Slide 9: A. Double strand DNA 50º Taq Taq Slide 10: Taq Taq E. Copy strands First round of cDNA synthesis (4 strands) Taq Taq Slide 11: 1 2 3 4 50º G. Anneal primers Slide 12: 1 2 3 4 72º H. Polymerase binds Slide 13: 1 2 3 4 Taq Taq Taq Taq I. Copy strands 72º Second round of cDNA synthesis (8 strands) Slide 14: 1 2 3 4 J. Denature at 96º Anneal primers at 50º Slide 15: 1 2 3 4 72º K. Bind polymerase (not shown) and copy strands Third round of cDNA synthesis (16 strands) Slide 16: 1 2 3 4 L. Denature at 96º Anneal primers at 50º Slide 17: 1 2 3 4 M. Copy strands at 72º Fourth round of cDNA synthesis (32 strands) 72º Slide 18: 1 2 3 4 cDNA strands (32) are now shown as lines Slide 19: 1 2 3 4 After 5 rounds there are 32 double strands of which 24 (75%) are are same size Post PCR step in Conventional PCR : Post PCR step in Conventional PCR Slide 21: In-Vitro amplification In-Vivo replication In-vivo replication : In-vivo replication Semi-conservative mode of replication DNA polymerase mediated No non-specific bindings At normal body temperature Real time RT-PCR : Real time RT-PCR Ø Uses conventional RT followed by PCR with florescent reporter dye and quencher. Specifically hybridize to the amplicon emits the fluorescence. Directly proportional to the target nucleic acid present. Advantages of Real Time -PCR : Advantages of Real Time -PCR Amplification, detection and quantitation of PCR product occur simultaneously in the same reaction vessel. Monitors the fluorescence emitted during the reaction in each cycle. Ø Allows convenient quantitation. Eliminates post-PCR manipulation, traditional gel electrophoresis. Ø Reduces carry over contamination. Ø Sensitivity reached up to 100 copies / ml. Ø More accurate quantification. Applications of PCR : Applications of PCR Human Health and diagnosis Biotechnology applications- Animal and Plant genetic engineering (Cloning) Tool for DNA sequencing Forensic and law- DNA finger printing Genetics Anthropological Hybridisation Probe Chemistry introduced by C.Wittwer : Hybridisation Probe Chemistry introduced by C.Wittwer Biotechnological : Biotechnological Recombinant DNA technology: DNA formed after a piece from one organism is joined to a piece from another organism (Cloning Vector). Inserted DNA is expressed in vector, multiplied and gives clones. Eg: Humulin (Eli-Lilly’s)-1982. Transgenic microbes, organisms (Plant and Animal) Multiplication of Geneproducts- Proteins, Glycoproteins etic.,, Environmental biotechnology- Eg.Pseudomonas putida Human Health : Human Health An essential tool for improving human health and human life. Two areas: Detection of infectious disease organisms and detection of variations and mutations in genes, especially human genes. Molecular diagnosis: PCR looks directly for the virus's unique DNA, So pathogens which are difficult or impossible to culture, such as many kinds of bacteria, fungi, and viruses, Eg: Detection of HIV, HCV, HBV, TB, SARS, Avian flu etc., sooner during the first few weeks after infection than the standard ELISA test (searching for antibodies the body has made against it). Molecular characterization of the disorder: Identifying the genetic change in the disorder. Eg. Cancer, Albinism, Autoimmune diseases, HPV, CMV etc., Basis for Human Genome Project (HGP): Identification and functional relation to its genes. Forensic, Genetic, Anthropological studies : Forensic, Genetic, Anthropological studies An indispensable adjunct to forensic DNA typing-commonly called DNA fingerprinting. "Ancient DNA" and Evolutionary Relationships : Genetic lineages: PCR is also helping sort out relationships among vanished human groups, and tracing human migrations. Slide 30: Genetic relationships Linkages Homology studies : Use of PCR in Forensic Science : OPEN HOUSE You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.
NANDU PCR Presentation nnandu1 Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 294 Category: Science & Tech.. License: All Rights Reserved Like it (0) Dislike it (0) Added: September 07, 2010 This Presentation is Public Favorites: 0 Presentation Description No description available. Comments Posting comment... By: dr.eshanidewan (10 month(s) ago) Dear Sir, I would like to have Quantitative RT-PCR presentation. Can you please send me the presentation to my mail ID eshanidewan@yahoo.com. I will be thankful to you. regards. eshani Saving..... Post Reply Close Saving..... Edit Comment Close By: jassu15747 (11 month(s) ago) sir how to download it...????? Saving..... Post Reply Close Saving..... Edit Comment Close By: nnandu1 (18 month(s) ago) Sorry for delay, You can download the presentation Saving..... Post Reply Close Saving..... Edit Comment Close By: sepideh (19 month(s) ago) Please allow me to download it! Thank you! Saving..... Post Reply Close By: nnandu1 (16 month(s) ago) Sorry for delay, no massage is posted You can download and feel free in comments nanda gopal Saving..... Edit Comment Close By: yekula (19 month(s) ago) Hello sir......... Could you please allow me to download this ppt Saving..... Post Reply Close Saving..... Edit Comment Close loading.... See all Premium member Presentation Transcript POLYMERASE CHAIN REACTION(PCR)&APPLICATIONS N.Nanda GopalJr.Scientist : POLYMERASE CHAIN REACTION(PCR)&APPLICATIONS N.Nanda GopalJr.Scientist Overview : Overview Differences between In-vivo and In-vitro replication Basics of PCR: Definition, invention. Updates of PCR: Available techniques and applications. Principle of PCR Components of PCR and their significance. Reaction steps with description. Applications: Industrial Human Health Agricultural- Plant & animal Forensic Genomic Invention of PCR : Invention of PCR Invented by Kary Banks Mullis (1983), Cetus corp, USA. Used klenow fragment of DNA polymerase-I, Major disadvantage - sensitive to high temps > 900C. Replaced by Lawyer et.al. (1989) with Taq DNA Polymerase of Thermus aquaticus, a volcanic achaebacteria. Thermostable, high fidelity with proof reading function were identified, Eg: Vent polymerase- Thermococcus litoralis Pfu polymerase- Pyrococcus furiosus Kary Mullis : Kary Mullis Born in a farmer family 1944, North Carolina, USA. Worked as Pharmaceutical Chemist at Cetus Corp., Nobel prize in Chemistry 1993. Japan Prize. Thomas A. Edison Award. Also invented UV-sensitive plastic that changes color in response to light. Technique : Technique In-Vitro, exponential amplification of target DNA sequences Using Taq-Polymerase (obtained from Thermus aquaticus), Primers- To initiate and to avoid non-specific DNA amplification flanked either side of the target. By controlled cycling the temperature, the target DNA is repetitively denatured and copied. A single copy of the target DNA can be amplified to obtain billions of replicates. To amplify RNA sequences, must be converted to DNA via reverse transcriptase. This two-phase procedure is known as ‘RT-PCR’. Components of PCR & significance : Components of PCR & significance Nucleiotides (dNTPs)- Building blocks Primers set (Forward & Reverse)- Initiation DNA Polymerase enzyme- Polymerization MgCl2- Co-factor for DNA Polymerase PCR Buffer- Stabilization of the reaction mix PCR Mix : PCR Mix Basic steps of PCR : Basic steps of PCR Thermal denaturation of the ds DNA at 940C (30 sec) Annealing of primers to their complementary sequences 50-600C(30sec) Extension at 720C (5 min) of the annealed primers with DNA polymerase. (Earlier was provided in different water baths) The amplified product is called “Amplicon”. Versatile technique with high speed, efficiency, sensitivity and specificity Eliminates the laborious conventional culture methods. Slide 9: A. Double strand DNA 50º Taq Taq Slide 10: Taq Taq E. Copy strands First round of cDNA synthesis (4 strands) Taq Taq Slide 11: 1 2 3 4 50º G. Anneal primers Slide 12: 1 2 3 4 72º H. Polymerase binds Slide 13: 1 2 3 4 Taq Taq Taq Taq I. Copy strands 72º Second round of cDNA synthesis (8 strands) Slide 14: 1 2 3 4 J. Denature at 96º Anneal primers at 50º Slide 15: 1 2 3 4 72º K. Bind polymerase (not shown) and copy strands Third round of cDNA synthesis (16 strands) Slide 16: 1 2 3 4 L. Denature at 96º Anneal primers at 50º Slide 17: 1 2 3 4 M. Copy strands at 72º Fourth round of cDNA synthesis (32 strands) 72º Slide 18: 1 2 3 4 cDNA strands (32) are now shown as lines Slide 19: 1 2 3 4 After 5 rounds there are 32 double strands of which 24 (75%) are are same size Post PCR step in Conventional PCR : Post PCR step in Conventional PCR Slide 21: In-Vitro amplification In-Vivo replication In-vivo replication : In-vivo replication Semi-conservative mode of replication DNA polymerase mediated No non-specific bindings At normal body temperature Real time RT-PCR : Real time RT-PCR Ø Uses conventional RT followed by PCR with florescent reporter dye and quencher. Specifically hybridize to the amplicon emits the fluorescence. Directly proportional to the target nucleic acid present. Advantages of Real Time -PCR : Advantages of Real Time -PCR Amplification, detection and quantitation of PCR product occur simultaneously in the same reaction vessel. Monitors the fluorescence emitted during the reaction in each cycle. Ø Allows convenient quantitation. Eliminates post-PCR manipulation, traditional gel electrophoresis. Ø Reduces carry over contamination. Ø Sensitivity reached up to 100 copies / ml. Ø More accurate quantification. Applications of PCR : Applications of PCR Human Health and diagnosis Biotechnology applications- Animal and Plant genetic engineering (Cloning) Tool for DNA sequencing Forensic and law- DNA finger printing Genetics Anthropological Hybridisation Probe Chemistry introduced by C.Wittwer : Hybridisation Probe Chemistry introduced by C.Wittwer Biotechnological : Biotechnological Recombinant DNA technology: DNA formed after a piece from one organism is joined to a piece from another organism (Cloning Vector). Inserted DNA is expressed in vector, multiplied and gives clones. Eg: Humulin (Eli-Lilly’s)-1982. Transgenic microbes, organisms (Plant and Animal) Multiplication of Geneproducts- Proteins, Glycoproteins etic.,, Environmental biotechnology- Eg.Pseudomonas putida Human Health : Human Health An essential tool for improving human health and human life. Two areas: Detection of infectious disease organisms and detection of variations and mutations in genes, especially human genes. Molecular diagnosis: PCR looks directly for the virus's unique DNA, So pathogens which are difficult or impossible to culture, such as many kinds of bacteria, fungi, and viruses, Eg: Detection of HIV, HCV, HBV, TB, SARS, Avian flu etc., sooner during the first few weeks after infection than the standard ELISA test (searching for antibodies the body has made against it). Molecular characterization of the disorder: Identifying the genetic change in the disorder. Eg. Cancer, Albinism, Autoimmune diseases, HPV, CMV etc., Basis for Human Genome Project (HGP): Identification and functional relation to its genes. Forensic, Genetic, Anthropological studies : Forensic, Genetic, Anthropological studies An indispensable adjunct to forensic DNA typing-commonly called DNA fingerprinting. "Ancient DNA" and Evolutionary Relationships : Genetic lineages: PCR is also helping sort out relationships among vanished human groups, and tracing human migrations. Slide 30: Genetic relationships Linkages Homology studies : Use of PCR in Forensic Science : OPEN HOUSE