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Isothermal amplification reagents

[NIPPONGENE] Thermostable Strand-Displacement DNA Polymerase

This product is an enzyme with 5' → 3' DNA polymerase activity and strand displacement activity, synthesizing a new DNA strand by releasing itself from the hydrogen bonds of double-stranded DNA that is used as a template. Thermostable strand-displacement DNA polymerases, which require no dissociation of double-stranded DNA due to their characteristics, can synthesize DNA at a constant temperature without being inhibited by the secondary structure of the DNA.

  • Features
    • 5' → 3' DNA polymerase activity and strand displacement activity
    • Able to synthesize DNA at a constant temperature
    • Lineup of three enzymes that differ in reaction temperature
    • Most suitable for synthesizing DNA strands with high GC content
  • Use
    • Application based on strand displacement activity
      (e.g., isothermal amplification)

Product lineup

Product name Optimal temperature Deactivation temperature*1 Component
BST DNA Polymerase 60~65℃ 80℃, 5 min 1) BST DNA Polymerase (8units/μl)
2) 10× BST Reaction Buffer (80mmol/l Mg2+)
1,600units
500μl
Csa DNA Polymerase 60~70℃ 85℃, 5 min 1) Csa DNA Polymerase (8units/μl)
2) 10× Csa DNA Polymerase (80mmol/l Mg2+)
1,600units
500μl
96-7 DNA Polymerase 50~55℃ 70℃, 5 min 1) 96-7 DNA Polymerase (8units/μl)
2) 10× 96-7 DNA Polymerase (95mmol/l Mg2+)
1,600units
500μl

*1 Inactivation temperature when enzyme stock solution is directly heat denatured.

Bst DNA Polymerase≫ Application for LAMP

LAMP reaction conditions

  • Bst DNA Polymerase
    Reaction: 65°C, 60 min

    <Composition of reaction mixture>

    Candidatus Liberibacter asiaticus DNA※2 1×106copies
    FIP 40pmol
    BIP 40pmol
    F3 Primer 5pmol
    B3 Primer 5pmol
    Loop Primer F 20pmol
    Loop Primer B 20pmol
    dNTPs Mixture 1.4mM each
    10× Bst Reaction Buffer 2.5 μl
    Bst DNA Polymerase 8units
    Total 25μl

    *2 The LAMP primer set for detection of Candidatus Liberibacter asiaticus was developed by the Kyushu Okinawa Agricultural Research Center of the National Agriculture and Food Research Organization under a project titled "Development of technology to prevent the spread of pesticide-resistant citrus greening disease," one of their projects promoting agricultural forestry and fisheries research using advanced technology.

  • <Lane>

    M:Gene-Ladder Wide 1(Product Number 313-06961)
    1 : Company A Bst DNA Polymerase
    2 : NIPPONGENE Bst DNA Polymerase

    <Note>

    3.0% agarose 21/TAE gel electrophoresis
    Ethidium bromide staining

Primer sequence

FIP: 5'-GCATGCCGAGGATCAATGCCTTGCTTAAAGAGCGTGCTACG-3'
BIP: 5'-TATGCCTAATGGCACGGGGGTAAGCTTCATCCGCCTTCGA-3‘
F3 Primer: 5'-TGGGTTAAGTGATGCTGTGG-3'
B3 Primer: 5'-CAACAATATCAGCCCCTGCT-3'
Loop Primer F: 5'-TCTCAACTGTTTCATCAAACCTAGC-3'
Loop Primer B: 5'- CGTGGCGGTTTTTGCTACA-3'

Csa DNA Polymerase / 96-7 DNA Polymerase≫ Application for LAMP

LAMP reaction conditions

<Composition of reaction mixture>

  • Csa DNA Polymerase
    Candidatus Liberibacter asiaticus DNA 1×106copies
    FIP 40pmol
    BIP 40pmol
    F3 Primer 5pmol
    B3 Primer 5pmol
    Loop Primer F 20pmol
    Loop Primer B 20pmol
    dNTPs Mixture 1.4mM each
    10× Csa Reaction Buffer
    Csa DNA Polymerase 8units
    total 25μl

    Reaction: 65°C, 60 min

  • 96-7 DNA Polymerase
    Candidatus Liberibacter asiaticus DNA 1×106copies
    FIP 40pmol
    BIP 40pmol
    F3 Primer 5pmol
    B3 Primer 5pmol
    Loop Primer F 20pmol
    Loop Primer B 20pmol
    dNTPs Mixture 2.0mM each
    10× 96-7 Reaction Buffer
    96-7 DNA Polymerase 8units
    total 25μl

    Reaction: 65°C, 60 min

Primer Sequence

  • FIP: 5'-GCATGCCGAGGATCAATGCCTTGCTTAAAGAGCGTGCTACG-3'
    BIP: 5'-TATGCCTAATGGCACGGGGGTAAGCTTCATCCGCCTTCGA-3‘
    F3 Primer: 5'-TGGGTTAAGTGATGCTGTGG-3'
    B3 Primer: 5'-CAACAATATCAGCCCCTGCT-3'
    Loop Primer F: 5'-TCTCAACTGTTTCATCAAACCTAGC-3'
    Loop Primer B: 5'- CGTGGCGGTTTTTGCTACA-3'

  • <Note>

    3.0% agarose 21/TAE gel electrophoresis
    Ethidium bromide staining

  • <Lane>

    M: Gene-Ladder Wide 1(Product Number 313-06961)
    1: Strand-displacement DNA polymerase derived from Geobacillus
    2: NIPPONGENE Csa DNA Polymerase
    3: NIPPONGENE 96-7 DNA Polymerase

Csa DNA Polymeras / 96-7 DNA Polymerase≫ Evaluation of thermostability and optimum temperature by RCA method

RCA reaction conditions

<Composition of reaction mixture>

  • M13mp18 single strand DNA 20ng
    Universal Primer 50nM
    dNTPs Mixture 0.25mM< each
    Tris-HCl (pH 8.8 at 25℃) 20mM
    KCl 10mM
    (NH4)2SO4 10mM
    MgSO4 2mM
    Tween 20 0.1%
    DNA Polymerase 8units
    total 20μl
  • <Lane>

    M: Gene-Ladder Wide 1(Product Number 313-06961)
    D: Template DNA(M13mp18) single strand DNA

Primer Sequence

Universal Primer
5'-GTTTTCCCAGTCACGACGTTGTA-3'

<Note>

3.0% agarose 21/TAE gel electrophoresis
Ethidium bromide staining

RCA(Rolling Circle Amplification) reaction diagram

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Isothermal Amplification-related Reagents

For research use or further manufacturing use only. Not for use in diagnostic procedures.

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