PCR/Amplification

Long & Accurate PCR

Many of today's PCR based applications require longer read lengths, greater fidelity and higher yields than that which can be achieved with Taq DNA polymerase. Cloning and expression experiments, cDNA analysis and array work can be improved by using long and accurate amplification over routine amplification.

Long and Accurate (LA) amplification is achieved by combining a thermostable polymerase with a second polymerase exhibiting a 3'—>5' exonuclease activity. The exonuclease, or proofreading, activity repairs terminal misincorporations. This repair allows for greater read lengths and increased fidelity.

Sigma Aldrich brings you a complete line of Long and Accurate products for DNA research. Our AccuTaq LA and KlenTaq LA enzymes and blends provide increased yield, higher processivity for greater lengths, and proofreading activity for increased fidelity over other long and accurate enzymes.


AccuTaq LA DNA Polymerase (D8045) and REDAccuTaq LA DNA Polymerase (D4812)

A blend of Sigma's quality Taq DNA polymerase with a 3'—>5' proofreading exonuclease. This mix provides increased yield and 6.5X greater fidelity over standard Taq DNA polymerase.

  • Generate amplicons up to 20 kb on complex, genomic templates or 40 kb on less complex DNA
  • Increased yield over competitors' long and accurate enzymes
  • REDAccuTaq allows for quick recognition of addition and mixing as well as direct loading to an agarose gel

 

 

Amplification of 25-40 kb lambda DNA template using AccuTaq LA DNA Polymerase
Lane 1: 25 kb
Lane 2: 31 kb
Lane 3: 36 kb
Lane 4: 40 kb
Lane M1: Lambda Hind III marker
Lane M2: PCR 100 bp ladder

Click here for more data on AccuTaq and REDAccuTaq LA DNA polymerase.


KlenTaq LA DNA Polymerase (D5062), KlenTaq DV ReadyMix (D1816), and RED KlenTaq DV ReadyMix (D1691)

An optimized mix of KlenTaq-1 and a proofreading exonuclease, these enzymes are the products of choice for amplifying DNA with difficult secondary structure or for applications requiring high yields and fidelity.

  • Generate amplicons up to 5 kb on complex templates or 20 kb on less complex DNA with higher fidelity (4X higher than standard Taq) using KlenTaq LA DNA polymerase
  • KlenTaq DV ReadyMix provides increased sensitivity and yield over Taq DNA polymerase master mixes
  • RED KlenTaq DV ReadyMix allows for quick recognition of addition and mixing as well as direct loading to an agarose gel
Amplification was performed with various amounts of lambda DNA using Taq DNA polymerase and KlenTaq LA DNA polymerase. Reaction volume was 50 5l and specific primers were chosen to generate 2.5 kb, 6 kb, 10 kb and 20 kb amplicaons.
Lane M: Marker
Lanes 1, 5: 2.5 kb amplicon from 1 ng starting material
Lanes 2, 6: 6 kb amplicon from 2 ng of starting material
Lanes 3, 7: 10 kb mplicon from 2.5 ng of starting material
Lanes 4, 8: 20 kb amplicon from 50 ng of starting materialA

Click here for more information on KlenTaq LA DNA polymerase and KlenTaq DV ReadyMixes


JumpStart AccuTaq LA DNA Polymerase (D5809) and JumpStart REDAccuTaq LA DNA Polymerases (D1313)

Combining an antibody based hot start mechanism with our long and accurate enzymes results in increased specificity and sensitivity to our high yield, high fidelity products. JumpStart AccuTaq is the ideal choice for high fidelity cloning applications or high yield array work.

  • Greater specificity with antibody inactivation over standard Taq or chemically inactivated enzymes
  • Hot start mechanism allows for room temperature set up of reactions, ideal for high throughput applications
  • Increased specificity, with amplification from as low as 0.4 ng of template

 

Long and accurate hot start enzymes were used to amplify a 5 kb fragment starting with 25 ng of total human genomic DNA. All reactions were performed according to the manufacturer's specifications.
Lane M: Wide Range DNA marker
Lane 2: JumpStart REDAccuTaq LA
Lane 3: Supplier S
Lane 4: Supplier I, enzyme P
Lane 5: Supplier I, enzyme HF

Click here for more information on JumpStart AccuTaq LA and JumpStart REDAccuTaq LA DNA polymerase


JumpStart KlenTaq LA DNA Polymerase (D0816)

An antibody inactivated formulation of KlenTaq LA delivers the high yield and improved processivity of KlenTaq with the specificity of a hot start enzyme.

  • Greater specificity with antibody inactivation over standard Taq or chemically inactivated enzymes
  • Increased yields compared to other hot start polymerases, especially when amplifying templates with high GC content

JumpStart KlenTaq LA and competitors' high performance enzymes were used to amplify an 890 bp amplicon containing 80% overall G-C content from total human genomic DNA. Reactions were run according to supplier's recommendations both without (Lanes 1-5) and with (Lanes 6-10) suggested PCR enhancers.

 

 

Run using supplier's recommended conditions
Lane M: PCR Marker
Lane 1: Sigma Taq
Lane 2: Supplier S, enzyme H
Lane 3: Supplier Q
Lane 4: Supplier S, enzyme Y
Lane 5: JumpStart KlenTaq LA

 

Run using supplier's recommended conditions and enhancers
Lane M: PCR Marker
Lane 6: Taq DNA polymerase witn 1M betaine
Lane 7: Supplier S, enzyme H with 4% DMSO
Lane 8: Supplier Q with 1X Q solution
Lane 9: Supplier S, enzyme Y with 4% DMSO
Lane 10: JumpStart KlenTaq LA with 1 M betaine