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Tutorials : Aug 1, 2008 ( )
Fast PCR with Long and Difficult Amplicons
TAQXpedite Was Designed to Reduce PCR Cycling Times in a Variety of Applications!--h2>
Fast PCR can be achieved with fast PCR instrumentation or by using enzyme/reagent combinations that improve speed and PCR efficiency. With a standard thermocycler, the TAQXpedite™ PCR System (Fast End-Point) from Epicentre Biotechnologies is three to four times faster than conventional cycling protocols.
The TAQXpedite PCR System contains a blend of thermostable DNA polymerases, an optimized 2X Universal MasterMix, and a Difficult/Long Master Mix with all four dNTPs. Both MasterMixes contain MgCl2 at optimized concentrations. The MasterMix also contains Epicentre’s PCR Enhancer, which substantially improves yield, efficiency, and specificity of amplification of many target sequences, especially those with a high GC content or secondary structure.
The Universal MasterMix is designed for routine and/or high-throughput fast PCR applications, while the Difficult/Long MasterMix is designed for use with amplifications that are difficult to achieve.
The data presented in this tutorial demonstrates that the TAQXpedite PCR System allows for rapid PCR cycling times with both routine and challenging templates. The time saving does not compromise PCR specificity or sensitivity.
Cycling Time Comparisons
To test the TAQXpedite System with a routine template, a 539 bp fragment was amplified from 1, 10, and 100 pg of lambda DNA using a standard thermocycler. Each reaction included 1X TAQXpedite Universal MasterMix, 12.5 pmol each of forward and reverse primers, and the specified amount of template. The cycling program was 98°C for 30 seconds, followed by 20 cycles: 92°C for 1 second and 65°C for 5 seconds.
Overall PCR time, comprising cycling and ramping time, was 16 minutes (Table). In contrast, a standard cycling procedure would require 1 hour and 16 minutes, nearly 80% more time. Agarose gel analysis of the PCR product showed highly specific amplification even with 1 pg of template (Figure 1).
To demonstrate that the TAQXpedite System can also be used for long PCR applications, 20 kb lambda and 30 kb fosmid sequences were amplified. Each reaction contained 1X TAQXpedite Universal MasterMix, 12.5 pmol each of forward and reverse primer, and 1 ng of template DNA. After an initial denaturation at 98°C for 1 minute, the reactions were amplified for 20 cycles: 98°C for 5 seconds (20 kb) or 10 seconds (30 kb), 60°C for 10 seconds (20 kb) or 30 seconds (30 kb), and 68°C for 7 minutes and 30 seconds (20 kb) or 15 minutes (30 kb).
The TAQXpedite Kit saved many hours in cycling time. The 20 kb lambda amplification was completed in just under three hours, while the 30 kb fosmid sequences were amplified in 5.5 hours. Standard cycling procedures for both lengths would require three- to fourfold more time. All PCR products were amplified with high specificity (Figure 2).
Fast Multiplex PCR
Amplifying several products in a single reaction often requires extensive optimization, because using multiple primer pairs increases the likelihood of forming nonspecific products. To evaluate the TAQXpedite System for multiplex amplification, nine regions of the human Duchenne muscular dystrophy (DMD) gene were amplified.
Each reaction contained 1X TAQXpedite Universal MasterMix, 12.5 pmol each of forward and reverse primer (nine pairs), and 50 ng of human genomic DNA. The cycling conditions were 98°C for 30 seconds, followed by 30 cycles: 98°C for 5 seconds, 58°C for 5 seconds, and 72°C for 30 seconds.
The nine-band multiplex PCR of the human DMD gene was successfully completed in 52 minutes with the TAQXpedite Universal MasterMix (Figure 3). No further optimization was required.
Fast PCR for High-GC Amplification
As with multiplex PCR, templates with high GC content or secondary structure are often challenging to amplify. To compare the TAQXpedite System with three other suppliers’ fast PCR kits, a 270 bp sequence that is 80% GC-rich was amplified from the human FMR1 (Fragile X) gene.
PCR amplifications were set up according to the manufacturer’s directions, including the reactivation of hot-start enzymes where applicable. Each reaction included 1X MasterMix, 12.5 pmol each of forward and reverse primers, and 50 ng of human genomic DNA. The cycling conditions were 98°C for 30 seconds, followed by 35 cycles: 98°C for 5 seconds and 68°C for 20 seconds.
Only the reaction containing TAQXpedite’s Difficult/Long MasterMix resulted in the desired 270 bp amplicon in 44 minutes. The other fast PCR systems yielded nonspecific products and a background smear (Figure 4).
Use of the TAQXpedite PCR System reduced PCR cycling times when compared to traditional cycling protocols. We have shown that routine PCR of a 500-bp fragment was completed in just 16 minutes. Improved cycling times were also achieved for a variety of challenging applications including amplification of templates 30 kb or greater in length, multiplex PCR, and amplification of GC-rich templates. Neither specificity nor sensitivity were compromised when the fast PCR format was used.
Haiying Grunenwald, Ph.D., is senior research scientist at Epicentre Biotechnologies. Web:www.epibio.com. Phone:(608) 442-6148.
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