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PCR01:32

PCR

Overview
DNA Isolation01:24

DNA Isolation

DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...

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Related Experiment Video

Updated: May 16, 2026

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1
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Isothermal quadruplex priming amplification for DNA-based diagnostics.

Adam Taylor1, Anupama Joseph, Robert Okyere

  • 1Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.

Biophysical Chemistry
|December 13, 2012
PubMed
Summary
This summary is machine-generated.

Quadruplex priming amplification (QPA) enables isothermal DNA amplification using a G3T sequence. The study reveals QPA

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Aptamer-Based Target Detection Facilitated by a 3-Stage G-Quadruplex Isothermal Exponential Amplification Reaction

Published on: October 6, 2022

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Quadruplex priming amplification (QPA) is a method for isothermal DNA amplification.
  • The assay utilizes a specific DNA quadruplex formed by the GGGTGGGTGGGTGGG (G3T) sequence.
  • G3T exhibits unique thermodynamic properties, influences quadruplex formation upon guanine removal, and facilitates fluorescent nucleotide emission.

Purpose of the Study:

  • To investigate the driving forces behind quadruplex priming amplification (QPA).
  • To determine the impact of 3'-terminal guanine removal on QPA efficiency.
  • To explore the optimal conditions and nucleotide incorporation characteristics of QPA.

Main Methods:

  • Model studies using primers with varying numbers of 3'-terminal guanines (missing one, two, or three).
  • Analysis of primer/template and product complex thermal stability (Tm).
  • Assessment of QPA efficiency at different temperatures and in the presence of specific dNTPs.
  • Evaluation of Taq polymerase incorporation opposite fluorescent nucleotides (2AP and 6MI).

Main Results:

  • QPA driving force is linked to the thermal stability difference between primer/template and product complexes.
  • Primers missing one or two 3'-guanines allow self-dissociation and support QPA.
  • QPA is not observed when three 3'-guanines are absent.
  • Optimal QPA rates occur slightly above the primer/template Tm and are enhanced with only dGTP.
  • Taq polymerase incorporates thymidines opposite template 2-aminopurine (2AP) but shows minimal incorporation opposite 6-methylisoxanthopterin (6MI).

Conclusions:

  • The thermal stability of primer-template interactions is crucial for QPA.
  • Specific primer designs (missing 1-2 3'-guanines) are necessary for efficient QPA.
  • QPA conditions can be optimized for maximum efficiency.
  • The behavior of Taq polymerase with fluorescent nucleotides provides insights into DNA polymerase fidelity and assay development.