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Related Experiment Videos

Nucleotide analogs facilitate base conversion with 3' mismatch primers.

J P Day1, D Bergstrom, R P Hammer

  • 1Department of Microbiology, Box 62, Hearst Microbiology Research Center, Strang Cancer Prevention Center, Joan and Sanford I. Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA.

Nucleic Acids Research
|April 2, 1999
PubMed
Summary

Nucleotide analogs, termed convertides, enable efficient DNA sequence conversion. This method effectively creates transition conversions, though transversions require further analog development.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Polymerase chain reaction (PCR) amplification is a cornerstone of molecular biology.
  • Modifying DNA sequences precisely is crucial for various genetic applications.
  • Nucleotide analogs offer potential for altering DNA synthesis outcomes.

Purpose of the Study:

  • To evaluate the efficiency of PCR amplification using primers with nucleotide analogs at the 3' base.
  • To investigate the ability of specific nucleotide analogs ('convertides') to induce sequence conversions.
  • To compare the efficacy of nucleotide analogs in facilitating transition versus transversion conversions.

Main Methods:

  • PCR amplification using primers with nucleotide analogs or mismatches at the 3' end.

Related Experiment Videos

  • Sequencing of PCR products to analyze base insertion opposite analogs.
  • Ligation detection reaction using sequence-specific primers to identify conversion products.
  • Comparison of conversion efficiency with and without a preconversion step.

    • Main Results:

    • Identified 'convertides' as nucleotide analogs enabling efficient amplification and base insertion.
    • Demonstrated successful conversion of C, T, G, and A bases into other bases using convertides.
    • Found transition conversions to be more efficient than transversions.
    • Observed that sequence context and primer slippage influence conversion outcomes and artifact formation.
    • Showed that nucleotide analogs can reduce artifacts and increase desired product yield.

    Conclusions:

    • Nucleotide analogs ('convertides') are effective tools for targeted DNA sequence conversion, particularly for transitions.
    • Primer slippage is a significant factor in artifact generation during analog-mediated conversion.
    • Further development of nucleotide analogs is necessary for reliable transversion conversions.
    • The study highlights the potential of analog-mediated PCR for genetic manipulation and sequence alteration.