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Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

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Formation of template-switching artifacts by linear amplification.

Dhrubajyoti Chakravarti¹, Paula C Mailander

¹Eppley Institute for Research in Cancer and Allied Diseases, 986805 Nebraska Medical Center, Omaha, NE 68198-6805, USA. dchakrav@unmc.edu

Journal of Biomolecular Techniques : JBT

|January 13, 2009

Summary

Linear amplification of DNA is efficient with single-stranded templates. Double-stranded templates produce artifact DNA products due to template switching, especially at higher concentrations.

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Area of Science:

Molecular Biology
Genetics

Background:

Linear amplification synthesizes single-stranded DNA using DNA primers, multiple DNA synthesis rounds, and thermostable DNA polymerases.
This method is generally more efficient with single-stranded DNA templates compared to double-stranded DNA templates.

Purpose of the Study:

To analyze the efficiency and fidelity of linear amplification using single-stranded versus double-stranded mouse H-ras DNA (exon 1-2 region).
To identify and characterize artifact products generated during linear amplification of double-stranded DNA templates.

Main Methods:

Linear amplification was performed on both single-stranded and double-stranded mouse H-ras DNA (exon 1-2).
Template concentrations were varied for the double-stranded template experiments.
Artifact DNA bands were sequenced to determine their origin.

Main Results:

Linear amplification of single-stranded H-ras DNA yielded only the intended full-length product.
Linear amplification of double-stranded H-ras DNA produced additional artifact products.
Higher concentrations of double-stranded template increased the relative amount of artifact products.
Sequencing revealed artifact products contained template-switching events, often associated with hairpin structures.

Conclusions:

Linear amplification is highly efficient and accurate with single-stranded DNA templates.
Double-stranded DNA templates are prone to generating artifact products through template switching during linear amplification.
Understanding these artifacts is crucial for accurate DNA synthesis and analysis using linear amplification.