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OF PHARMACEUTICS NCP PAMBADY 13–06-2O11CONTENTS: CONTENTS INTRODUCTION PRINCIPLE OF PCR THE “REACTION” COMPONENTS TEHNIQUES OF PCR VARIANTS OF PCR APPLICATIONS OF PCR ADVANTAGES OF PCR DISADVANTAGES OF PCR CONCLUSION REFERENCES PCR: PCR The polymerase chain reaction ( PCR ) is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence . 3 It was invented in 1983 by Dr. Kary Mullis, for which he received the Nobel Prize in Chemistry in 1993.WHY “POLYMERASE”?: WHY “POLYMERASE”? 4 It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase. WHY “CHAIN”?: WHY “CHAIN”? 5 It is called “chain” because the products of the first reaction become substrates of the following one, and so on.PRINCIPLE OF PCR: PRINCIPLE OF PCR Double stranded DNA is denatured into two individual strands ( Denaturation) Each strand is allowed to hybridise with a primer ( Renaturation ) Primer – template duplex is used for DNA synthesis – enzyme DNA polymerase ( Synthesis ) 3 steps are repeated again and again to generate multiple forms of target DNA 6 THE “REACTION” COMPONENTS: THE “REACTION” COMPONENTS Target DNA - the sequence to be amplified (100 – 1000bp) Two primers - complementary to the DNA target( 17 – 30 nucleotides) DNA polymerase - Tma DNA polymerase ( Thermotoga maritama), Pfu DNA polymerase ( Pyrococcus furiosus ) 4 dNTPs Buffer solution - providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. Divalent cation - magnesium ions Monovalent cation - potassium ions Promoters - bovine serum albumin Inhibitors - humic acid 8THE REACTION: THE REACTION 9 THERMOCYCLER PCR tubeTECHNIQUES OF PCR: TECHNIQUES OF PCR Initialization step : - Heating the reaction to a temperature of 94–96 °C - held for 1–9 minutes. -required for DNA polymerases that require heat activation Denaturation step : - heating the reaction to 94–98 °C for 20–30 seconds. - causes melting of the DNA template - By disrupting the hydrogen bonds between complementary bases 10Slide 11: Annealing step : - The reaction temperature is lowered to 50–65 °C for 20–40 seconds - allowing annealing of the primers to the single stranded DNA template Extension/Elongation step : - DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand - By adding dNTPs that are complementary to the template in 5' to 3' direction - condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent DNA strand. - Extension time and temperature depends DNA polymerase used and length of the DNA fragment to be amplified 11STAGES OF PCR: STAGES OF PCR 3 stages: Exponential : -The amount of product is doubled - 100% reaction efficiency - The reaction is very sensitive Levelling off(Linear) stage : - The reaction slows due to lose of activity of DNA polymerase and consumption of reagents such as dNTPs and primers. Plateau : - No more product accumulates due to exhaustion of reagents and enzyme. 13Slide 14: 14VARIANTS OF PCR : VARIANTS OF PCR INVERSE PCR ANCHORED PCR R EVERSE T RANSCRIPTION PCR (RT-PCR) ASYMMETRIC PCR NESTED PCRANCHORED PCR: ANCHORED PCR Small sequence of nucleotides attached to target DNA (poly G tail) Useful when sequence surrounding the target DNA is not known 16INVERSE PCR : INVERSE PCR Amplification of DNA of unknown sequence from known sequence 17NESTED PCR: NESTED PCR 2 pairs of primers used First pair of PCR primers amplify a fragment similar to standard PCR Second pair of primers (nested primers) bind inside the first PCR product to allow amplification of second PCR product Specific PCR amplification 18REVERSE TRANSCRIPTION PCR (RT-PCR) : R EVERSE T RANSCRIPTION PCR (RT-PCR) For amplifying DNA from RNA. Reverse transcriptase reverse transcribes RNA into cDNA, which is then amplified by PCR 19ASYMMETRIC PCR: ASYMMETRIC PCR - Synthesis of single stranded DNA molecules - Used in sequencing and hybridization probing where amplification of only one of the two complementary strands is required - Use great excess of the primers for the chosen strand 20APPLICATIONS OF PCR: APPLICATIONS OF PCR Classification of organisms Genotyping Molecular archaeology Mutation detection Sequencing Cancer research Sex determination of embryos Detection of pathogens DNA fingerprinting Drug discovery Genetic matching Genetic engineering Pre-natal diagnosis Forensic medicine 21Slide 22: M OLECULAR IDENTIFICATION 22DETECTION OF UNKNOWN MUTATIONS: DETECTION OF UNKNOWN MUTATIONS 23Slide 24: SSCP gels: “shifts” representing a mutation in the amplified DNA fragment 24DETECTION OF PATHOGENS: DETECTION OF PATHOGENS 25 Prenatal diagnosis of inherited disease - Sickle Cell Anemia - Thalassemia - Phenyl Ketonuria Diagnosis of retroviral infections Diagnosis of bacterial infections - Tuberculosis Diagnosis of cancers - Cervical cancer – Papilloma virusSlide 26: Detection of PCR-amplified M. tuberculosis DNA. 26DNA SEQUENCING: 27 DNA SEQUENCINGFORENSIC MEDICINE: FORENSIC MEDICINE 28 blood, chorionic villus, amniotic fluid, semen, hair root, saliva 68,719,476,736 copies Identification of criminals ADVANTAGES OF PCR: ADVANTAGES OF PCR Minute amount of starting material Quick and simple Extremely sensitive Robustness 29DISADVANTAGES OF PCR: DISADVANTAGES OF PCR Chance of contamination Theoretically one molecule can amplify Need for target DNA sequence information Short size and limiting amounts of PCR product Small size range of the DNA sequences that can be cloned – In cell-based DNA cloning where the size of cloned DNA sequences can approach 2 Mb but DNA sequences cloned by PCR have typically been in the 0.1–5 kb size range Infidelity of DNA replication 30CONCLUSION: CONCLUSION 31 The speed and ease of use, sensitivity and specificity of PCR made PCR the most widely used and powerful technique with great spectrum of research and diagnostic applications . REFERENCES: REFERENCES 1. U.Sathyanarayana,Biochemistry P.111 - 119 2. http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/ TechPCR.shtml 3. http://users.ugent.be/~avierstr/principles/pcr.html 4. http://en.wikipedia.org/wiki/Applications_of_PCR 5. http://en.wikipedia.org/wiki/Inverse_polymerase_chain_reaction 6.http://www.pcrstation.com/nested-pcr/ 7.http://catalog.takara-bio.co.jp/en/product/basic_info.asp?unitid=U100005940 8. http://catalog.takara-bio.co.jp/en/product/basic_info.asp?unitid=U100005356Slide 33: THANK YOU You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.