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  • Acyclonucleotide Set

    Description

    Acyclonucleotide Set contains four separate tubes of acyNTPs (acyATP, acyCTP, acyGTP and acyTTP).
    Acyclonucleotides (acyNTPs) act as chain terminators and are thus useful in applications that normally employ dideoxynucleotides such as DNA sequencing (1,2) and SNP detection (3). AcyNTPs are especially useful in applications with archaeon DNA Polymerases, more specifically with Therminator™ DNA Polymerase. Therminator DNA Polymerase is an engineered enzyme with an increased capacity to incorporate analogs with altered sugars, such as ribonucleotides, dideoxynucleotides, 2´ deoxynucleotides and especially acyclo-base analogs (4,5).

    When used with Therminator DNA Polymerase (NEB #M0261), the concentration of acyclo-base analog is roughly equivalent to dideoxynucleotide concentrations used with Thermo Sequenase™ (see figure).

    (50 ng/µl single-stranded M13mp18 DNA, 0.05 µM [32P]-S1224S primer, 1X ThermoPol Buffer, 50 µM dNTP, varying acyGTP and 0.05 units/µl Therminator) were mixed, incubated at 94°C for 5 minutes and then thermal cycled for 25 cycles at 94°C (30 seconds); 55°C (30 seconds); 72°C (30 seconds). Reaction products were separated on a denaturing polyacrylamide gel (QuickPoint, NOVEX, Inc.) and analyzed by autoradiography. Numbers above each lane give the molar ratio of acyGTP to dNTP in each lane (i.e., 50, 17, 5,6 and 1.9 µM acyGTP, respectively). Numbers to the side of the gel indicate the length of the primer extension product added to the primer.

    Properties and Usage

    Storage Temperature

    -20°C

    Notes

    1. Thermo Sequenase™ is a trademark of GE Healthcare.
    2. Addition of 50 μl of distilled or deionized (Milli-Q™) water will result in a final concentration of 10 mM acyNTP. The hydrated acyNTP solution should subsequently be stored at -20°C.

    References

    1. Sanger, F., Nicklen, S. and Coulson, A.R. (1977). Proc. Natl. Acad. Sci. USA. 74, 5463-5467.
    2. Trainor, G.L. (1996). U.S. Patent No. 5,558,991.
    3. Haff, L.A. and Simirnov, I.P. (1997). Genome Methods. 7, 378-388.
    4. Gardner, A.F. and Jack, W.E. (1999). Nucl. Acids Res.. 27, 2545-2553.
    5. Gardner, A.F. and Jack, W.E. (2002). Nucl. Acids Res.. 30(2), 605-613.

    FAQs

    1. Are acyclonucleotides chain terminators?
    2. Are substrates terminated with 3' acyclonucleosides resistant to 3'-5' exonuclease activity?
    3. Which DNA polymerases incorporate acyclonucleotides?
    4. Can acyclonucleotides be used in Sanger sequencing?
    5. Can acyclonucleotides be used for sequencing-by-synthesis?

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

    Interactive Tools

    Quality Control

    Quality Assurance Statement

    • Acyclonucleotide solutions are certified free of nucleases and phosphatases.  Functional purity is determined by chain termination sequencing reactions.

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
    • Functional Test (Sanger Sequencing):
      Functional purity is tested in Sanger chain terminator sequencing reactions using Therminator DNA Polymerase.

    Safety Data Sheet

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.