VeraSeq ULtra DNA Polymerase

OEM by QIAGEN offers bulk manufacturing of VeraSeq ULtra DNA Polymerase in custom formulations.

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VeraSeq ULtra DNA Polymerase (500 U)

Cat. No. / ID: P7520L

500 U (evaulation pack) of VeraSeq ULtra DNA Polymerase, 5X VeraSeq Buffer II and 5X VeraSeq GC Buffer.
This product is licensed from Bio-Rad Laboratories, Inc., under U.S. Pat. Nos. 6,627,424,7,541,170,7,560,260 and corresponding patents in other countries for use solely in DNA sequencing, DNA micro-array, and conventional PCR applications, including pre-amplification steps that are required for such applications, in the life science research and in-vitro diagnostics fields but not real-time PCR or digital PCR.  
The VeraSeq ULtra DNA Polymerase is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Features

  • Read and use uracil residue with maximum speed and accuracy
  • Ultra-thermostable polymerase
  • 25x greater fidelity than Taq DNA polymerase
  • Functions in Mg2+ concentrations 1.5 to 3.0 mM
  • Strong proofreading function (3'-5' exonuclease)

Product Details

VeraSeq ULtra DNA Polymerase is an engineered, ultra-thermostable, uracil-literate polymerase designed to maximize the speed, accuracy, and length of DNA synthesis during sequencing template preparation. The result is a novel enzyme that can read through uracil, extend a kilobase of sequence in 15 seconds and has an accuracy 25 times higher than Taq DNA Polymerase.

 

VeraSeq ULtra DNA Polymerase is supplied in 20 mM Tris-HCl, 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, stabilizer and 50% glycerol; pH 7.4 at 25°C.

 

VeraSeq ULtra DNA Polymerase is provided with 5X VeraSeq Buffer II (B7102) and 5X VeraSeq GC Buffer (B7130).

 

Ask about low-glycerol or hot-start formulations.

Performance

Polymerase properties
Extension rate: 15 seconds per kilobase at 72˚C
Proofreading (3'-5' exo): Yes, strong
Nick-translation (5'-3' exo): No
Fidelity: > 25X higher than Taq DNA Polymerase
Strand displacement: No
Thermostability: Highly thermostable
Able to extend an RNA primer: No
Extends from a nick: No
Generate blunt end products: Yes
Uracil read through: Yes

Polymerase properties

  • Storage temperature: –25°C to –15°C
Test Units tested Specification
Purity n/a > 95%
Specific activity n/a 100,000 U/mg
Double-stranded endonuclease 120 U No conversion
E. coli DNA contamination 150 U <10 copies

 

Principle

Source of recombinant enzyme protein
The protein is produced by a recombinant E. coli strain carrying the engineered VeraSeq ULtra gene.

Unit definition:
One unit is defined as the amount of enzyme required to incorporate 10 nmoles of dNTPs into acid-insoluble form at 74°C in 30 minutes.

Procedure

Protocol
General precautions should be taken when setting up PCR, including setting up the reaction on ice, adding the polymerase last, gentle pipetting, thorough mixing and a quick centrifugation. The following procedure can be used as a guideline. Reactions may need to be optimized individually.

Reaction setup (for 50 µl)

Component  Volume (µl) Final concentration
Sterile H2O Variable n/a
5X VeraSeq buffer II or 5X VeraSeq GC buffer ULtra 1X
10 mM dNTP mix 1 200 µM each
Primer 1 Variable 0.2 µM
Primer 2 Variable 0.2 µM
DNA template Variable See note 4, below
VeraSeq  ULtra DNA Polymerase 0.5 1 U

Total reaction volume can be adjusted as needed


Typical cycling conditions

Step Temperature Time Cycles
Initial denaturation  98°C 30 seconds 1
Denaturation
Annealing
Extension
98°C
Varies
72°C
5–10 seconds
10–30 seconds
15–30 seconds/kb
15–35
Final extension  72°C
4°C
5–10 min
Hold
1
Cycling conditions may need to be optimized, depending on the amplicon of interest

Notes:

1. 5X VeraSeq Buffer II should be used as the default buffer for high-fidelity amplification. For GC-rich and difficult templates, use 5X VeraSeq GC Buffer
2. VeraSeq 2.0 ULtra DNA Polymerase can be used in PCR amplification to generate deoxyuridine-containing products by including dUTP in the reaction. If DNA template contains uracil or dUTP needs to be incorporated, use VeraSeq Ultra (P7520L) 
3. A final concentration of 0.2 µM is recommended for each primer, but it can be varied in the range of 0.2–1 µM     
4. Recommended template quantities:

Complexity Source example Guideline
Low Plasmid, virus, BAC 1 pg – 10 ng
High Genomic DNA 50–250 ng

     5. One unit is usually sufficient for amplifying most targets. For long targets (>1kb), difficult templates or to increase yield, it may be necessary to add up to 2 units of enzyme.

     6. Both 5X VeraSeq Buffer II and GC buffer are formulated to provide a final 1X concentration of MgCl2 of 1.5 mM. In cases where additional Mg2+ is required, adjust the final Mg2+ concentration in 0.2 mM steps. 

     7. For GC rich templates, DMSO may be used to reduce the secondary structure of complex templates. DMSO is generally used at a 3% final concentration (v/v). If additional optimization is required, adjust the concentration in 1–2% increments (2–9% in final reaction). The primer annealing temperature should be lowered to account for the presence of the solvent.

     8. VeraSeq ULtra DNA Polymerase is also compatible with other PCR-enhancing additives, such as BSA and betaine.

Quality control analysis

Specific activity was measured using a twofold serial dilution method. Dilutions of enzyme were made in 1X reaction buffer and added to 50 µl reactions containing activated calf thymus DNA; 25 mM TAPS (tris-[hydroxymethyl]-methyl-amino-propanesulfonic acid, sodium salt) pH 9.3 at 25°C; 50 mM KCl; 2 mM MgCl2; 1 mM β-mercaptoethanol; 200 µM each dATP, dGTP, dTTP; and 100 µM [3H]-dCTP (0.075 Ci/mmole). Reaction vessels were mixed and incubated at 74°C for 10 minutes.

Protein concentration is determined by OD280 absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the band corresponding to the protein of interest in the diluted sample.

Double-stranded endonuclease activity is determined in a 50 µl reaction containing 0.5 µg of plasmid DNA and 10 µl of enzyme solution incubated for 4 hours at 37°C.

E. coli contamination is evaluated using 5 µl replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

Applications

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

  • High-fidelity DNA amplification
  • Cloning
  • Synthetic biology

Resources

FAQ

When should I use 5X VeraSeq GC Buffer?
VeraSeq GC Buffer is recommended for use with difficult or GC-rich amplicons. GC Buffer may also improve yield of some targets. VeraSeq GC Buffer is also recommended for extended room temperature incubation of VeraSeq ULtra DNA polymerase with reaction components prior to PCR cycling. 
FAQ ID - 3923
How is VeraSeq ULtra DNA Polymerase different from the standard recombinant Taq-B DNA Polymerase?
VeraSeq ULtra DNA Polymerase has higher fidelity, speed, and performance compared to Taq-B DNA Polymerase. VeraSeq ULtra DNA Polymerase has an error rate that is 25 times lower than Taq-B DNA Polymerase and can extend 1 kb of sequence per 15 seconds, drastically reducing cycling times. VeraSeq ULtra DNA Polymerase produces blunt end products whereas Taq-B leaves a single-base 3’ overhang. 
FAQ ID - 3921
What is the fidelity/error rate of VeraSeq ULtra DNA Polymerase?
VeraSeq ULtra DNA Polymerase has an error rate of 1.0 x 10-6 in VeraSeq Buffer II and 7.3 x 10-7 in VeraSeq GC Buffer when measured using a LacI-based assay (2).
FAQ ID - 3925
How can yield for targets be increased when using VeraSeq ULtra DNA Polymerase?
Use of a PCR enhancer containing Betaine, DTT, BSA, and DMSO (3) may also be used to improve yield of complex targets (especially targets ≥ 3 kb). Increasing the extension time, the template concentration, or polymerase concentration to 2 U/50 µL may also be helpful. 
FAQ ID - 3926
Do the DNA fragments generated by VeraSeq ULtra DNA Polymerase have a single-base 3´ overhang?
No. VeraSeq ULtra DNA Polymerase generates blunt end products. 
FAQ ID - 3928
What annealing temperature should be used in the cycling conditions?
Set the annealing temperature approximately 3°C higher than the lowest Tm primer for oligos greater than 20 nucleotides. For oligos shorter than 20 nucleotides, set the annealing temperature equal to the Tm of the lowest Tm primer.  When the Tm of the primer pairs is ≥ 72°C, use two-step cycling conditions where the annealing and extension steps are combined. If DMSO is needed in the reaction, a reduction of the annealing temperature is often necessary. 
FAQ ID - 3932
What denaturation temperature should be used in the cycling conditions?
VeraSeq ULtra DNA Polymerase is highly thermostable and it is recommended that denaturation should be performed at 98°C. The initial denaturation time can be extended from 30 seconds up to 3 minutes for difficult templates. For subsequent denaturation cycles, 5-10 seconds at 98°C is sufficient for most targets. 
FAQ ID - 3931
What is the amplification length limit of VeraSeq ULtra DNA Polymerase?
VeraSeq ULtra DNA Polymerase has been demonstrated to amplify up to 5 kb of human genomic DNA and up to 8 kb for lambda DNA.  
FAQ ID - 3924
How can I optimize Mg2+ conditions for a specific amplicon when using VeraSeq ULtra DNA Polymerase and the supplied reaction buffers?
VeraSeq Buffer II and VeraSeq GC buffer result in a final concentration of 1.5 mM Mg2+ in the reaction. Therefore, final Mg2+ reaction concentration may be increased according to user preference with a concentrated solution containing Mg2+.  VeraSeq ULtra DNA Polymerase works under a broad range of Mg2+ concentrations (1.5 – 3.0 mM) but higher Mg2+ can compromise fidelity (1).     
FAQ ID - 3922
Will VeraSeq ULtra DNA Polymerase incorporate dUTP?
Yes.
FAQ ID - 3927
Is VeraSeq ULtra DNA Polymerase available as a hot start enzyme?
No. Currently VeraSeq ULtra DNA Polymerase is not available with a hot start function as a standard product.
FAQ ID - 3929