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tools for Molecular Biology Amplification

tools for Molecular Biology Amplification. The PCR Reaction. The PCR reaction is a way to quickly drive the exponential amplification of a small piece of DNA. PCR is a 3 step process Denaturation of the target DNA Annealing of your gene specific primers

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tools for Molecular Biology Amplification

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  1. tools for Molecular BiologyAmplification

  2. The PCR Reaction The PCR reaction is a way to quickly drive the exponential amplification of a small piece of DNA. PCR is a 3 step process • Denaturation of the target DNA • Annealing of your gene specific primers • Elongation of the target DNA by a Heat Stabile DNA Polymerase • Amplification progresses exponentially so that the final number of copies equals 2n (n=number of cycles)

  3. 5’ 3’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 3’ 5’ 3’ The PCR Reaction d.NTPs Primers Add Master Mix and Sample Thermal Stable DNA Polymerase Add to Reaction Tube Denaturation Annealing

  4. 5’ 5’ 5’ 3’ 3’ 3’ 5’ 5’ 3’ 3’ 3’ 3’ 5’ 5’ 3’ Taq Taq 5’ 5’ 5’ Taq Taq 5’ The PCR Reaction Extension Extension Continued Repeat

  5. 3’ 3’ 5’ 5’ 5’ 3’ 3’ 3’ 5’ 3’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ THE PCR REACTION Cycle 2 4 Copies Cycle 3 8 Copies

  6. PCR - Powerful Tool!! • PCR technology is an essential tool for Molecular Biology • PCR allows rapid and reproducible amplification of a specific sequence of DNA • PCR technology is responsible for accelerating Genetic Discoveries

  7. What is Real Time PCR? Real Time PCR incorporates the ability to directly measure and quantify the reaction while amplification is taking place.

  8. Real Life Threshold Reality vs. Theory Amplification is exponential, but the exponential increase is limited: • A linear increase follows exponential • Eventually plateaus Theoretical Log Target DNA Real-Time PCR allows us to ‘see’ the exponential phase so we can calculate how much we started with. CT Cycle #

  9. Threshold Cycle, Ct, of the same 96 replicates shows nearly identical values

  10. CT What is Threshold Cycle (CT)?

  11. The threshold cycle, Ct • Correlates strongly with the starting copy number • If you have twice the template, you get to Ctone cycle earlier • If you have half the template, you reach Ctone cycle later

  12. Threshold Cycle, CT Detection of 125 genomic equivalents from 250.Two-fold serial dilutions of human genomic DNA (gDNA) from 125 to 16,000 genomic equivalents were assayed for b-actin.

  13. The slope of the standard curve can be directly correlated to the efficiency of the reactions: Efficiency (h) = [10(-1/slope) ] - 1 Threshold Cycle, CT, can be used to generate standard curves r = is a measure of how well the actual data fit to the standard curve. = (explained variation/total variation)

  14. Threshold Cycle, Ct, is a reliable indicator of initial copy number

  15. What Detection Strategies can we use?

  16. Intercalating Dyes • Intercalating Dyes are inexpensive compared to hybridization probes. • - general confirmation of amplification - NON SPECIFIC • Russ Higuchi demonstrated the key principle of Real Time PCR using Ethidium Bromide - • EtBr fluoresces 25 times more brightly when bound to dsDNA • SYBR Green, a more sensitive intercalating dye is an even more attractive approach • SYBR Green fluoresces 200 times more brightly when bound to dsDNA

  17. 5’ 3’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ Taq ID 5’ 3’ 5’ 3’ Intercalating Dyes d.NTPs Primers Intercalation Dyes Add Master Mix and Sample Thermal Stable DNA Polymerase Reaction Tube l Denaturation Annealing

  18. 5’ 5’ 5’ 3’ 3’ 3’ 5’ 5’ 3’ 3’ 3’ 3’ 5’ 5’ 3’ Taq Taq 5’ 5’ 5’ Taq Taq l l l ID ID ID ID ID ID ID ID ID ID 5’ l l Intercalating Dyes Extension Extension Continued Apply Excitation Wavelength Repeat

  19. Hybridization Probes Today Hybridization Probe Strategiesfall into three main categories: • Cleavage Based Assay • TaqManä Assays • Displaceable Probe Assays • Molecular Beacons • Dual oligo FRET probes • Probes incorporated directly into the primers • Amplifluor • Scorpions

  20. Q 5’ 3’ R 5’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ Taq Probe 5’ 3’ Q R 5’ 3’ 5’ 3’ 5’ 3’ TaqManTM d.NTPs Primers Add Master Mix and Sample Thermal Stable DNA Polymerase Reaction Tube Denaturation l Annealing

  21. Q R 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ R R Q 3’ Taq Taq Taq Taq 5’ 3’ Q R R R 5’ 3’ 3’ 5’ Q Q 3’ 5’ TaqManTM Extension Step 1. Strand Displacement 2. Cleavage 3. Polymerization Complete l 4. Detection

  22. 5’ 3’ 5’ 3’ d.NTPs 3’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ Molecular Beacon Primers Add Master Mix and Sample 5’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ Thermal Stable DNA Polymerase Taq R Q Denaturation Q R 5’ 3’ 5’ 3’ 5’ 3’ Molecular Beacons Reaction Tube Annealing

  23. 5’ 5’ 5’ 3’ 3’ 3’ Q R 3’ 5’ 5’ 5’ 3’ Molecular Beacon Taq Taq R Q 3’ 5’ Q R 5’ 3’ Molecular Beacons l Detection Extension Step 1. Strand Displacement 2. Polymerization Complete Probe Silent

  24. 5’ 5’ 5’ 3’ 3’ 3’ R R R R D D D 3’ 5’ 5’ 5’ 3’ Taq Taq 3’ 5’ 3’ 5’ FRET Probes l Detection 1-5 bases Extension Step 1. Strand Displacement System Silent 2. Polymerization Complete System Silent

  25. Q Q R R 5’ 3’ Primer Based l Heat Incorporation l

  26. 3’ Annealing/Extension 1 5’ 5’ 3’ Q Q Q R R R 5’ 3’ 5’ 5’ 3’ 3’ 5’ Primer Based Extension 2 l Detection

  27. Melt Curve Analysis • This type of analysis measures the decrease in fluorescence as the temperature slowly increases. • The decrease in fluorescence is caused by the probe dissociating from the target. • Since changes in sequence result in changes in Tm, one can detect mutations by comparing the amount of fluorescence observed at different Tm. • Mutation detection (SNPs) is not the only application for Melt Curve Analysis • Probe or primer and target melt characterization, and validation of reactions with SYBR Green are other popular uses for Melt Curve Analysis.

  28. Melt Curve: what is it? • Discriminates by Melting Temperature (Tm) - the temperature at which 50% of the DNA molecules separate into two strands - or “melts” apart • Tm is dependent on: • sequence (G/C content) • length • complementarity

  29. change in fluorescence Tm single amplified product Fluorescence vs. Temperature

  30. Validation with SYBR Green Yes We Still Run Gels!!!

  31. Melt CurveCheck specificity of the reaction Melt curve showing two amplified products

  32. Melt Curve • Tests for specificity in all samples • Discriminates by melting temperature • Not as high resolution as running a gel • Run melt curve followed by gel of representative samples

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