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Variability in the pattern of random amplified polymorphic DNA

D K Khandka1, M Tuna, M Tal

  • 1The Albert Katz Center for Desert Agrobiology, The Jacob Blaustein Institute for Desert Research, Ben-Gurion University of the Negev, Sede Boker Campus, Israel.

Electrophoresis
|March 21, 1998
PubMed
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The random amplified polymorphic DNA (RAPD) technique is sensitive to reaction conditions, impacting DNA polymorphism detection. Optimizing reactant concentrations is crucial for reliable amplification patterns and accurate genetic marker analysis.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • The random amplified polymorphic DNA (RAPD) technique is widely used for DNA polymorphism detection.
  • RAPD is known for its sensitivity to reaction conditions, which can affect amplification outcomes.

Purpose of the Study:

  • To investigate how variations in reaction constituents influence RAPD amplification patterns.
  • To identify key factors affecting the reliability and reproducibility of RAPD markers.

Main Methods:

  • Examined DNA amplification variability using samples from Asparagus officinalis, Dactylis glomerata, Mercurialis annua, and Escherichia coli.
  • Systematically altered concentrations of Taq DNA polymerase, primers, and magnesium.
  • Assessed the impact of DNA fragmentation methods (sonication, restriction enzymes) on amplification.

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Main Results:

  • Taq DNA polymerase to DNA ratio influenced the number of amplified fragments.
  • Primer concentration affected the molecular weight of amplified DNA fragments.
  • Magnesium concentration was critical for the intensity of specific amplified fragments.
  • DNA fragmentation methods (mechanical or enzymatic) altered amplification patterns, leading to loss or gain of products.

Conclusions:

  • RAPD technique's sensitivity to reaction conditions necessitates careful optimization for accurate DNA polymorphism analysis.
  • Variations in reactant concentrations and DNA integrity significantly impact RAPD marker reliability.
  • Pooled DNA RAPD markers require critical evaluation due to potential 'false positive' results.