Applied Biosystems™ Sequenase Version 2.0 DNA Polymerase

The Sequenase™ Version 2.0 DNA Polymerase is a genetically engineered variant of T7 DNA polymerase, optimized for DNA sequencing and other molecular biology applications. It features enhanced functionality, including high processivity, strand displacement synthesis, and the ability to incorporate nucleotide analogs, making it an excellent choice for applications like dideoxy DNA sequencing. The enzyme has virtually no 3'→5' exonuclease activity, which minimizes degradation of DNA during reactions, ensuring high-quality and precise results.

Key Features

1. Genetic Engineering for Optimized Functionality:
   • Modified T7 DNA polymerase with a deletion of 28 amino acids that eliminates measurable 3'→5' exonuclease activity while preserving polymerase activity.
2. High Processivity:
   • Efficiently synthesizes long stretches of DNA without dissociating from the template.
3. Nucleotide Analog Incorporation:
   • Supports incorporation of nucleotide analogs such as dlTP, thio-dNTPs, and dideoxy-NTPs.
4. Strand Displacement Synthesis:
   • Effective for synthesizing complementary DNA strands in the presence of secondary structures.
5. High Purity:
   • Purity >95% as determined by SDS-PAGE, free from contaminating endonucleases and exonucleases.

Enzyme Properties

• Subunit Composition:
  • Modified T7 gene 5 protein: 76 kDa.
  • E. coli thioredoxin: 12 kDa.
• Optimum pH: 7.5.
• Optimum Temperature: 37°C.
• Divalent Cation Requirement: Requires Mg²⁺ or Mn²⁺ for activity.

Storage Information

• Storage Buffer:
  • 20 mM potassium phosphate (pH 7.4), 1 mM DTT, 0.1 mM EDTA, 50% glycerol.
• Storage Conditions:
  • Store at -20°C to maintain enzyme stability.

Applications

1. Dideoxy DNA Sequencing:
   • Particularly suited for Sanger sequencing due to its ability to incorporate dideoxy-NTPs and nucleotide analogs.
2. High-Precision DNA Synthesis:
   • Ideal for applications requiring minimal degradation of DNA.
3. Strand Displacement Assays:
   • Performs efficient synthesis even in templates with secondary structures.
4. Experimental Use:
   • Useful in research workflows requiring engineered DNA polymerase with no exonuclease activity.

Assay Conditions

• Standard Reaction Conditions:
  • Reaction mixture (100 μL):
    • 40 mM Tris-HCl (pH 7.5).
    • 10 mM MgCl₂.
    • 5 mM DTT.
    • 0.3 mM dNTPs.
    • 5 μg M13mp18 template pre-annealed with 5 pmol M13 universal primer.
  • Enzyme is added to the pre-warmed mixture and incubated at 37°C for 1 minute.
• Unit Definition:
  • One unit catalyzes the incorporation of 1 nmol of nucleotide into acid-insoluble form in 30 seconds at 37°C.

Included Components

1. Enzyme Concentration:
   • 13 units/μL of Sequenase Version 2.0 DNA Polymerase.
2. 5X Sequenase Reaction Buffer (1 mL):
   • 200 mM Tris-HCl (pH 7.5), 100 mM MgCl₂, 250 mM NaCl.
   • Product number: PN 70702.
3. Sequenase Dilution Buffer (1 mL):
   • 10 mM Tris-HCl (pH 7.5), 5 mM DTT, 0.1 mM EDTA.
   • Product number: PN 70765.

Functional Testing

The enzyme is functionally tested using the Sequenase Version 2.0 DNA Sequencing Kit (PN 70770), ensuring its effectiveness in DNA sequencing applications.

Advantages

1. Exonuclease-Free Activity:
   • Ideal for applications where degradation of template DNA must be avoided.
2. Compatibility with Nucleotide Analogs:
   • Supports versatile applications in sequencing and mutagenesis studies.
3. Highly Reliable and Pure:

   • 95% purity ensures consistent and reproducible results in sensitive molecular biology workflows.

4. Strand Displacement:
   • Facilitates efficient synthesis even in structurally complex DNA templates.

Applications in Research

1. Sanger Sequencing:
   • Accurate dideoxy sequencing with reduced interference from secondary structures.
2. Mutagenesis Studies:
   • Incorporation of analog nucleotides to study DNA modifications.
3. Strand Displacement Assays:
   • Effective for synthesizing complementary strands over complex DNA regions.
4. High-Fidelity DNA Amplification:
   • Ensures error-free replication for downstream applications.

This engineered DNA polymerase is a highly versatile and reliable enzyme for advanced molecular biology workflows, making it a preferred choice for sequencing and research requiring high precision and processivity.

References

• Tabor, S. and Richardson, C. C. (1989) J. Biol. Chem. 264, 6447-6458.
• Wang, D., et al. (2002) Proc. Natl. Acad. Sci. USA, 99, 15687-15692.
• Tabor, S. and Richardson, C. C. (1987) Proc. Natl. Acad. Sci. USA 84, 4767-4771.
• Paris, M. (1992) United States Biochemical Corporation Comments 18, No. 3.