PCR PRODUCTS How to Select the Best PCR Enzyme How To Select the Best PCR Enzyme 1 2 www.takara-bio.eu Introduction to Takara’s Patented LA PCR Technology Ex Taq™ Polymerase offers high sensitivity, increased product yield and length, and improved reproducibility and fidelity over Taq Polymerase. LA Taq™ Polymerase can synthesize products up to 48 kb in length with fidelity 6.5X better than Taq Polymerase, and requires less optimization than other long PCR polymerases. LA Taq™ Polymerase with GC Buffer is optimized for amplification of GC-rich templates, and SpeedSTAR™ HS DNA Polymerase is highly processive, with reaction times up to 5X faster than Taq. Premix and Hot-Start versions of Ex Taq™ and LA Taq™ are available, as well as qPCR, RT-PCR, RACE, cloning, mutagenesis, and screening kits. PCR PRODUCTS Taq Polymerase, the workhorse enzyme most commonly used for PCR, has several limitations. It does not possess a “proofreading” (3’g5’ exonuclease) activity, and so has a relatively high rate of base misincorporations (error rate). At the end of a typical 30-cycle PCR reaction, a significant portion of the products generated with Taq Polymerase will contain one or more errors, especially in longer products. Because Taq Polymerase tends to “fall off” DNA templates at the sites of these misincorporations, both product yield and length are restricted. Long and Accurate PCR Technology (LA PCR) provides a solution to the problems intrinsic to the use of conventional Taq Polymerase. LA PCR Technology involves mixing Taq Polymerase with a small amount of a proofreading polymerase, producing an enzyme mix with performance characteristics (fidelity, yield, length, reproducibility) superior to either enzyme alone. Takara Bio owns the worldwide patent on Long and Accurate (LA) PCR, and provides the largest available selection of high-performance PCR reagents and kits based on this technology. 1 How To Select the Best PCR Enzyme Principle of LA PCR Technology. The key to LA PCR technology is the enzyme mix. Both Takara Ex Taq™ and Takara LA Taq™ are thermostable DNA polymerases which possess 3’g5’ exonuclease, or proofreading activity. This 3’g5’ exonuclease activity removes misincorporated bases, allowing subsequent product extension to proceed smoothly and efficiently, making amplification of long DNA fragments possible. www.takara-bio.eu 3 PCR PRODUCTS Guide to TaKaRa Application Polymerase* Amplification Efficiency Product Size w/ λDNA (Average/Max) TaKaRa Taq™* (R001A) ++ 6 kb/12 kb Premix Taq™ (R004A) ++ 6 kb/12 kb TaKaRa Taq HS* (R007A) ++ 6 kb/12 kb Premix Taq™ HS (R028A) ++ 6 kb/12 kb EmeraldAmp® GT PCR Master Mix* (RR310A) ++ 6 kb/12 kb TaKaRa Ex Taq™* (RR001A) ++++ 20 kb/30 kb Premix Ex Taq ™ (RR003A) ++++ 20 kb/30 kb TaKaRa Ex Taq™ HS* (RR006A) ++++ 20 kb/30 kb Premix Ex Taq ™ HS (RR030A) ++++ 20 kb/30 kb PerfectShot™ Ex Taq (RR005A) ¥ ++++ 20 kb/30 kb EmeraldAmp® Max PCR Master Mix* (RR320A) ¥ ++++ 20 kb/30 kb EmeraldAmp® Max HS PCR Master Mix* (RR330A) ¥ ++++ 20 kb/30 kb PrimeSTAR MAX* (R045A) ++++ up to 6 kb PrimeSTAR GXL* (R050A) ++++ 30 kb PrimeSTAR HS* (R010A) +++ up to 20 kb PrimeSTAR HS with GC Buffers (R044A) +++ up to 10 kb PrimeSTAR HS, Premix (R040A) +++ up to 10 kb TaKaRa LA Taq™* (RR002A) +++ 35 kb/48 kb TaKaRa LA Taq™ w/GC Buffers (RR02AG) +++ 35 kb/48 kb§ LA PCR Kit, V.2.1 (RR013A) +++ 35 kb/48 kb One-Shot LA PCR Mix V.2.0 (RR004) +++ 35 kb/48 kb TaKaRa LA Taq™ HS* (RR042A) +++ 35 kb/48 kb SpeedSTAR™ HS* (RR070A) +++ 20 kb/30 kb SapphireAmp® Fast PCR Master Mix* (RR350A) ¥ +++ 20 kb/30 kb SYBR Premix DimerEraser ™* (RR091A) +++ _ SYBR Premix Ex Taq™*(Tli RNaseH Plus) (RR420A) ++++ _ SYBR Premix Ex Taq™* II (Tli RNaseH Plus) (RR820A) ++++ _ Premix Ex Taq™*(Probe qPCR) (RR390A) ++++ _ Routine PCR Guide to Takara Polymerases 1 High Efficiency PCR ® ® High Fidelity PCR ® ® ® Long PCR Fast PCR ® ® Real Time PCR ® g * Sample Available Unit Definition One unit is the amount of enzyme that will incorporate 10 nmol of dNTP into acid-insoluble products in 30 min. at 74°C with activated salmon sperm DNA as the template-primer. Purity Nicking activity, endonuclease, and exonuclease activity were not detected after the incubation of 0.6 µg of double-stranded supercoiled pBR322 DNA, 0.6 µg of λ DNA, or 0.6 µg of λ-Hind III digest with 10 units of enzyme for 1 hour at 74°C. 4 www.takara-bio.eu PCR Polymerases Product Size w/ Human Genomic DNA (Average/ Max) Hot Start PCR 2 kb/4 kb Processing Speed Terminal Transferase Activity (3’-A overhang) ++ + _ 1 kb/min Yes No ++++ + _ 1 kb/min Yes +** No ++ + +++ 1 kb/min Yes Yes +** No ++++ + +++ 1 kb/min Yes 2 kb/4 kb No +** No ++ +++ _ 1 kb/min Yes 10 kb/20 kb No ++** Yes ++ + _ 1-2 kb/min Yes 10 kb/20 kb No ++** Yes ++++ + _ 1-2 kb/min Yes 10 kb/20 kb Yes ++** Yes ++ + ++ 1-2 kb/min Yes 10 kb/20 kb Yes ++** Yes ++++ + ++ 1-2 kb/min Yes 10 kb/20 kb No ++** Yes ++ + _ 1-2 kb/min Yes 10 kb/20 kb No ++** Yes ++ ++ _ 1-2 kb/min Yes 10 kb/20 kb Yes ++** Yes ++++ ++ _ 1-2 kb/min Yes up to 6 kb Yes ++++ Yes +++ +++ _ 1 kb/5-10 sec No (blunt end) 30 kb Yes +++ Yes ++++ ++++ _ 1 kb/10 sec No (blunt end) up to 8.5 kb Yes ++++# Yes ++ +++ _ 1-2 kb/min No (blunt end) up to 5 kb Yes ++++# Yes ++ ++++ _ 1-2 kb/min No (blunt end) up to 5 kb Yes ++++# Yes ++++ +++ _ 1-2 kb/min No (blunt end) 20 kb/30 kb No +++ ** Yes ++ + _ 1-2 kb/min Yes+ (20 kb/30 kb)§ No +++ ‡** Yes ++ ++++ _ 1-2 kb/min Yes+ 20 kb/30 kb No +++** Yes ++ ++++ _ 1-2 kb/min Yes+ 20 kb/30 kb No +++** Yes ++++ + _ 1-2 kb/min Yes+ 20 kb/30 kb Yes +++** Yes ++ + _ 1-2 kb/min Yes+ 10 kb/ 20 kb Yes ++** Yes +++ + _ 6 kb/min Yes 10 kb/ 20 kb Yes ++** Yes +++ ++ _ 6kb/min Yes _ Yes ++** Yes ++++ + ++++ _ Yes _ Yes ++** Yes ++++ + ++++ _ Yes _ Yes ++** Yes ++++ + ++++ _ Yes _ Yes ++** Yes ++++ + ++++ _ Yes Fidelity Robustness No +** No 2 kb/4 kb No +** 2 kb/4 kb Yes 2 kb/4 kb § When used with GC Buffer I. ‡ When amplifying GC-rich templates, the fidelity is reduced. ** All fidelity determined by using the Kunkel method. # Fidelity determined by direct sequencing. www.takara-bio.eu 1 ++++Best +++Good ++Average +Poor 5 Guide to Takara Polymerases *A ll of Takara’s PCR polymerases are provided with dNTPs and buffer. They guarantee low DNA enzyme contamination (≤ 10 fg). + T-vector cloning efficiency diminishes as the length of the PCR product to be cloned increases above 5 kb. ¥ Dye added "Load N Go" Premixes. PCR PRODUCTS GC-Rich Templates Real Time PCR (qPCR) Proofreading Activity