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Primer-Free Aptamer Selection Using A Random DNA Library
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Optimized quantitative PCR analysis of random DNA aptamer libraries.

Keenan Pearson1, Caroline Doherty2, Drason Zhang3

  • 1Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, 200 First St. SW, Rochester, MN, 55905, USA; Biochemistry and Molecular Biology Track, Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic College of Medicine and Science, 200 First St. SW, Rochester, MN, 55905, USA.

Analytical Biochemistry
|May 13, 2022
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Summary
This summary is machine-generated.

Standard quantitative PCR (qPCR) can fail for random DNA libraries. Adjusting fluorescence monitoring detects annealed regions, enabling accurate analysis of these essential in vitro selection tools.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Quantitative polymerase chain reaction (qPCR) is crucial for nucleic acid analysis.
  • Standard qPCR methods face challenges with random DNA libraries used in in vitro selection.
  • Intercalator fluorescence detection of duplex DNA yield is common but can be unreliable for specific library types.

Purpose of the Study:

  • To investigate the failure of conventional qPCR methods in quantitating random DNA libraries.
  • To identify the underlying causes of unexpected results in qPCR analysis of these libraries.
  • To propose simple solutions for reliable automated analysis of random DNA libraries using qPCR.

Main Methods:

  • Experimental analysis of quantitative polymerase chain reaction (qPCR) performance.
  • Investigation of PCR product renaturation dynamics in late cycles.
  • Monitoring intercalator fluorescence at varied temperatures and time points.
  • Comparison of fluorescence detection based on full duplex DNA versus annealed constant regions.

Main Results:

  • Incomplete renaturation of PCR products containing random regions occurs in late cycles due to reagent depletion.
  • Extension failure in late PCR cycles leads to loss of intercalator fluorescence at standard interrogation temperatures.
  • Adjusting the fluorescence monitoring step to detect annealed constant regions, not full duplex DNA, resolves qPCR analysis issues.
  • This adjustment enables accurate qPCR analysis of random DNA libraries.

Conclusions:

  • Conventional qPCR methods can fail for random DNA libraries due to incomplete renaturation and reagent depletion.
  • Modifying the qPCR protocol by monitoring fluorescence of annealed constant regions ensures reliable quantitation.
  • This simple adjustment allows for successful automated analysis of random DNA libraries, crucial for in vitro selection experiments.