• My NEB
  • Print
  • PDF
  • 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

    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.
    • Functional Test (Sanger Sequencing):
      Functional purity is tested in Sanger chain terminator sequencing reactions using Therminator DNA Polymerase.

    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.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    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.
    1. Which thermophilic DNA polymerase should I use?
    2. What should I take into consideration when designing a set of PCR primers?
    3. How can I facilitate the amplification of templates with hairpin-loop structures or high GC-content?
    4. How important is the quality of my DNA template in long PCR?
    5. What kind of reaction tubes are recommended?
    6. What is two-step PCR?
    7. What if my primer extension reaction yields no product or a smear?
    8. What causes an occasional smear in a "negative control" with no template present?
    9. Can PCR products be phosphorylated in the PCR mixture?
    10. How can I improve blunt-end ligation efficiency of PCR products?
    11. What is the enzyme of choice for chewing back 3' overhangs and filling in 5' overhangs (3' recessed ends)?
    12. What is touchdown PCR?
    13. Which NEB DNA polymerases can incorporate fluorescently-labeled nucleotides during PCR?
    14. How can I amplify DNA from single cells?