Loop mediated isothermal amplification LAMP c Helicase dependent amplification

Loop mediated isothermal amplification lamp c

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Loop-mediated isothermal amplification (LAMP) c) Helicase-dependent amplification (HAD) d) Rolling circle amplification (RCA) e) Strand displacement amplification (SDA) PCR: 3 steps of PCR: 1. Denaturation: separation of the DNA strand @ 94 C 2. Annealing: 55-65 C Primers hybridize to the separated strands. DNAP always require primer to synthesize the nucleotides. Temperature depends on stringency required but generally set at Tm -5 C 3. Extension: synthesis of the new strand (72C) PCR cycle 1 and 2 after 35 th cycle, we have 34 billion copies. Detecting amplicons: 2 ways a. Conventional PCR: end of the rxn (end-point analysis) GEL LECTROPHORSIS b. Real-time PCR (qPCR): during the rxn FLUORESCENT DYE. Types of PCR: Conventional PCR Real-time (quantitative) PCR (q-PCR) Modification of either: Hot-start PCR Nested PCR Multiplex PCR Reverse transcriptase PCR (RT-PCR) Random Amplified Polymorphic DNA (RAPD) PCR 1. Hot start PCR: is to prevent non-specific amplification during the set up of PCR rxn by inactivating the Taq polymerase at low temperatures. DNAP is inactive, heat then activate DNAP
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2. Nested PCR: 2 sets of primers, use two separate rounds of PCR to increase the specificity and absorb sensitivity. 3. Multiplex PCR: adds many primers to the mixture of target genes to amplify at the same time, instead of doing separate PCR. Note: have to know the size of the target gene. 4. Reverse transcriptase PCR (50 degree C): amplify RNA by using reverse transcriptase to reverse trx. RNA to cDNA, then uses this cDNA for PCR. Components of a PCR rxn: Essential PCR buffer MgCl2 (1-4 mM)- is important for activity of Taq polymerase dNTP mix (200 uM each) Thermostable Polymerase Sterile deionized, RNAse/DNAse free water. Primer pairs Template DNA/RNA Optional Reverse transcriptase Fluorescent dyes Probes Video: 1. Wear clean PPE prior the experiment 2. all reagents should be kept on iced except reverse transcriptase and Taq polymerase 3. have the addition of positive and negative control of PCR. 4. Mix all PCR reagents in a big tube for making a master mix. 5. add fluorescent probe into he master mix 6. Keep master mix on ice to prevent the degradation of RNA and Taq polymerase 7. MgCl2 is used because thermostable polymerase requires the presence of magnesium to act as a cofactor during the reaction process. Its role is similar to that of a catalyst: the magnesium is not actually consumed in the reaction, but the reaction cannot proceed without the presence of the magnesium. Commonly used thermostable polymerases: o Taq polymerase from Thermus aquaticus , which is most commonly used. o Pfu polymerase is from Pyrococcus furiosus , high fidelity applications, minimize errors to minimum but more expensive than Taq polymerase. Have 2 domains….
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  • Summer '17
  • Al-Hebshi
  • DNA, RNA

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