tools for Molecular Biology Amplification

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 2 n (n=number of cycles) The PCR Reaction

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

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

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

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

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

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

Threshold Cycle, C t, of the same 96 replicates shows nearly identical values

CTCTCTCT What is Threshold Cycle (C T )?

n Correlates strongly with the starting copy number u If you have twice the template, you get to C t one cycle earlier u If you have half the template, you reach C t one cycle later The threshold cycle, C t

Threshold Cycle, C T 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  -actin.

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

Threshold Cycle, C t, is a reliable indicator of initial copy number

What Detection Strategies can we use?

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

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

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

Hybridization Probes Cleavage Based Assay – TaqManä Assays Displaceable Probe Assays – Molecular Beacons – Dual oligo FRET probes Probes incorporated directly into the primers – Amplifluor – Scorpions Today Hybridization Probe Strategies fall into three main categories:

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

5’ 3’ 5’3’ TaqMan TM 5’ 3’ R Q Extension Step 5’ 3’ 1. Strand Displacement Taq 3’ QR 5’ 3’ Q Taq R 5’ 2. Cleavage 3. Polymerization Complete 5’ 3’ Q Taq R 3’ 5’ 4. Detection 5’ 3’ Q Taq R 5’ R

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

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

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

Primer Based 3’ 5’ R Q Heat Incorporation R Q

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

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.

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

Fluorescence vs. Temperature change in fluorescence single amplified product TmTm

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

Check specificity of the reaction Melt Curve Check specificity of the reaction Melt curve showing two amplified products

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