Long and Accurate PCR

What is long and accurate PCR?

Many polymerase chain reaction (PCR) applications require longer read lengths, greater fidelity and higher yields than that which can be achieved with Taq DNA polymerase. Conventional Taq DNA polymerase lacks proofreading capability, resulting in a relatively high error rate. Taq polymerase also tends to disassociate from the template and cease elongation at sites of misincorporated nucleotides. Cloning and expression experiments, cDNA analysis and array work can be improved by using long and accurate amplification over conventional PCR.

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.

We offer products to achieve successful long and accurate PCR. Our AccuTaq LA enzyme and blends provide increased yield, higher processivity for greater lengths, and proofreading activity for increased fidelity over other long and accurate enzymes.

Increase PCR yield and fidelity

AccuTaq™ LA DNA Polymerase (Product No. D8045) and REDAccuTaq™ LA DNA Polymerase (Product No. D4812) are blends of our 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 other suppliers' long and accurate enzymes
  • REDAccuTaq™ LA DNA Polymerase enables 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 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

 

Achieve longer amplicons and greater yields with AccuTaq™ LA DNA polymerase. AccuTaq™ LA DNA polymerase (SA) and other suppliers' long and accurate enzymes were used to amplify 5 kb, 10 kb and 20 kb fragments of human genomic DNA.
 

AccuTaq™ LA DNA polymerase compared to other suppliers' long and accurate enzymes to amplify by PCR 5 kb, 10 kb and 20 kb fragments of human genomic DNA.

PCR amplification of beta-globin fragment
from human genomic DNA template using
AccuTaq™ LA DNA polymerase.
Amplification conditions:
5 kb amplifications: 50 µL, 5 µL were loaded onto the gel. For all samples 4 ng/µL of human genomic DNA was amplified with 0.05 units/µL of enzyme.
Cycling conditions: 94 °C 1 min, 30 cycles [94 °C 15 sec, 65 °C 20 sec, 72 °C 10 min] 72 °C 10 min final extension, hold at 4 °C
10 kb amplifications: 20 µL, 4 µL were loaded onto the gel. For all samples 1 ng/µL of human genomic DNA was amplified.
Enzyme amounts:
AccuTaq™ LA polymerase (SA): 0.05 units/µL
Supplier R: 0.075 units/µL
Supplier S: 0.1 units/µL
Cycling conditions: 94 °C 30 sec, 30 cycles [94 °C 5 sec, 62 °C 20 sec, 68 °C 20 min] 68 °C 20 min final extension, hold at 4 °C
15 kb amplifications: 20 µL, 4 µL were loaded onto the gel. For all samples 1 ng/µL of human genomic DNA was amplified.
Enzyme amounts:
AccuTaq™ LA polymerase (SA): 0.05 units/µL
Supplier R: 0.075 units/µL
Supplier S: 0.1 units/µL
Cycling conditions: 94 °C 30 sec, 30 cycles [94 °C 5 sec, 62 °C 20 sec, 68 °C 30 min] 68 °C 30 min final extension, hold at 4 °C

 

 

Product No. Product Name Features
D8045 AccuTaq LA DNA Polymerase • Amplify DNA sequences including genomic targets larger than 20 kb
• Increase fidelity up to 6.5X that of Taq DNA polymerase
D4812 REDAccuTaq™ LA DNA Polymerase • The inert red dye provides quick recognition and confirmation of appropriate mixing
• Efficiently and accurately produce amplicons up to 22 kb on genomic templates and up to 40 kb on less complex templates, such as lambda or bacterial DNA

Long and accurate antibody-based hot start PCR

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™ (Product No. D5809) and JumpStart™ REDAccuTaq™ LA DNA Polymerase (Product No. D1313) are ideal for high fidelity cloning applications or high yield array work.

  • Greater specificity with antibody inactivation over standard Taq polymerase 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
Electrophoresis of PCR products from combining an antibody-based hot start mechanism with our long-and-accurate enzymes, resulting in an increase in specificity and sensitivity.
Amplification of a 5 kb fragment using long and accurate hot start enzymes.
All reactions were performed according to the suppliers' recommendations using 25 ng of total human genomic DNA.
Lane M: Wide Range DNA marker
Lane 2: JumpStart™ REDAccuTaq™ LA DNA Polymerase
Lane 3: Supplier S
Lane 4: Supplier I, enzyme P
Lane 5: Supplier I, enzyme HF

 

Product No. Product Name Features
D5809 JumpStart™ AccuTaq™ LA DNA Polymerase • A combination of AccuTaq™ LA DNA Polymerase and a Taq-directed antibody
• Provides increased specificity and higher target yield in comparison to standard amplification
D1313 JumpStart REDAccuTaq® LA DNA Polymerase • Combines AccuTaq™ LA DNA polymerase and JumpStart™ Taq antibody with an inert red dye
• The inert red dye provides quick recognition and confirmation of appropriate mixing