Part 2 - Fast QPCR

Fast qPCR; The Why and How. Francisco Bizouarn Bio-Rad Laboratories www.bio-rad.com/genomics Contents ™Introduction ™Accelerating the Amplification Introduction www.bio-rad.com/genomics Definitions ™A Fast PCR Assay ™Assay designed to achieve desired amount of amplification product in a specific amount of time. ™A Fast qPCR Assay ™Assay designed to provide quantitative results within a desired level of accuracy in a specific amount of time. ™Usually requires about 40 min for products of <500 bp. www.bio-rad.com/genomics Why ™Is speed is a critical parameter? ™Am I willing to spend extra time optimizing? ™Am I willing to redesign the “old faithful” assay(s)? ™How much sensitivity do I need? www.bio-rad.com/genomics What makes a reaction faster? The qPCR InstrumentThe SupermixThe Driver Accelerating the Amplification www.bio-rad.com/genomics Classic PCR 3 min 1 min 1 min 7 min Activation Cycle 39Cycle 1 1 min Cycle 2 Cycle 40 Initial Ramp to 95o 40 X Ramp down to 60o 40 X Ramp up to 72o 39 X Ramp up to 95o 46 min total in ramping 3 min Polymerase Activation 40 min Denaturing 40 min Annealing 40 min Extension 7 min Final Extension 130 min total in incubations Total time 2 hours 56 minutes www.bio-rad.com/genomics Classic qPCR 3 min 1 min Activation Cycle 38Cycle 1 10 sec Cycle 2 Cycle 40Cycle 39 Initial Ramp to 95o 40 X Ramp down to 60o 39 X Ramp up to 95o 46 min total in ramping 3 min Polymerase Activation 7 min Denaturing 40 min Annealing 50 min total in incubations Total time 1 hour 36 minutes www.bio-rad.com/genomics Accelerating the process ™What can be done to accelerate the process even further? ™Time consuming components: ™Initial incubation to heat activate Taq ™Ramping down to annealing temp ™Annealing temp ™Ramping up to denaturing temp ™Denaturing temp www.bio-rad.com/genomics iTAQTM Activation 95deg 98deg 3min 1min 3min 1min 30s 15s 15 20 25 30 1 2 3 log Initial K562 targets per ul A v g C ( t ) 95 deg, 3 min 98 deg, 30 sec 10-31/11-1-0595 & 98 enz act plots (7) Log of starting quantity A v e r a g e C t www.bio-rad.com/genomics %G+C vs Tm & Tmax of various genomic DNAs 70 80 90 100 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 %G+C T m o r T m a x Tmax is the temperature at which all of the genomic DNA is denatured. Tm Tm & Tmax v Genomic GC ASCB (2) C.p. H.s. E.c. pBR322 M.l. Tm As of Dec. 1, 2005, only 3 sequenced genomes have GC contents of 70%GC or higher (highest 72.1) (www.genomesonline.org). About 300 of the sequenced genomes list a %GC . Initial denaturation of Genomic DNAs www.bio-rad.com/genomics Primer Tm determination Various Tm estimates from web-available Tm calculators for the sequence (ACGT)7 (Calculators not necessarily set at common concentration or thermodynamic properties.) 0 10 20 30 40 50 60 70 80 90 100 A B C D E F G H I J K L M N O P Q R S Tm calculators C a l c u l a t e d T m Average = 65.2 max = 79.0 min = 56.8 Tm histo (ACGT)7; 11-14-05 (2) www.bio-rad.com/genomics 98deg.C, 30sec; (92deg.C, 1sec; XXdeg.C, 15sec) X 35; 72deg.C, 1 min. Beta-Actin Beta-Globin M 58 60 62 64 66 68 70 72 M 58 60 62 64 66 68 70 72 M Average primer Tm 71 66 61 58 Primer Tm and fast 2-step PCR www.bio-rad.com/genomics Real Time Gradient 75oC 73.3oC 59.5oC 70.3oC 55oC 65.8oC www.bio-rad.com/genomics Robust Amp in short times All candidates mode in MyiQ, iQ and iQ5 1 min annealing/extension steps www.bio-rad.com/genomics Target denaturation Tm Tmax www.bio-rad.com/genomics Denaturation of Amplicons 70 75 80 85 90 95 100 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Amplicon %GC T m Tm &Tmax v %GC 150bp ASCB 3.0% GC change = 1deg. C change in Tm for 150bp amplicons Tmax Tm Tm vs %GC; 150bp ampliconsin Supermix+SYBR® Green 1 www.bio-rad.com/genomics Accelerating the process ™What can be done to accelerate the process even further? ™Time consuming components: ™Initial incubation to heat activate Taq Reduce to 30 sec at 98o ™Ramping down to annealing temp Anneal primers higher (70o) ™Annealing/Extension temp Reduce to 10 to 15 sec ™Ramping up to denaturing temp Lower to tested value (90o) ™Denaturing temp Reduce to 1 sec www.bio-rad.com/genomics Fast qPCR 30 sec 10 sec Activation Cycle 38Cycle 1 1 sec Cycle 2 Cycle 40Cycle 39 Initial Ramp to 98o 40 X Ramp down to 70o 39 X Ramp up to 92o 30 min total in ramping 30 sec Polymerase Activation 40 sec Denaturing 400 Sec Annealing/Extension 8 min total in incubations Total time 38 minutes www.bio-rad.com/genomics Fast Thermocycler Effect ™In addition to these time savings, can a fast thermocycler block reduce the assay time? ™Traditional block heats at 3.3o/sec and cools at 2o/sec. ™Fast blocks can heat at 6o/sec and cool at 4o/sec. ™All blocks require some time to reach max ramping rate and decelerate dramatically before reaching the target temperature so as not to over/undershoot the specified temperature. ™Using a “fast thermocycler” only reduces the assay time due to ramping by about 6 to 8 minutes. Somewhat! Fast qPCR; The Why and How. Contents Introduction Definitions Why What makes a reaction faster? Accelerating the Amplification Classic PCR Classic qPCR Accelerating the process iTAQTM Activation Initial denaturation of Genomic DNAs Primer Tm determination 98deg.C, 30sec; (92deg.C, 1sec; XXdeg.C, 15sec) X 35; 72deg.C, 1 min. Real Time Gradient Robust Amp in short times Denaturation of Amplicons Accelerating the process Fast qPCR Fast Thermocycler Effect