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Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis
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Dissecting and tuning primer editing by proofreading polymerases.

Daryl M Gohl1,2,3, Benjamin Auch1, Amanda Certano3

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|June 14, 2021
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Summary

Proofreading polymerases can edit PCR primers to improve DNA amplification accuracy, especially for microbiome profiling. This primer editing is tunable and can correct primer mismatches, rescuing data quality.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Proofreading polymerases possess 3' to 5' exonuclease activity for DNA replication error correction.
  • Previous work showed these polymerases can edit PCR primers to match template sequences, a process termed primer editing.
  • Primer editing offers potential advantages in applications like amplicon-based microbiome profiling.

Purpose of the Study:

  • To develop synthetic DNA standards for quantifying and dissecting primer editing activity.
  • To investigate the factors influencing primer editing, including polymerase concentration, cycling conditions, and sequence specificity.
  • To explore methods for tuning primer editing, such as incorporating phosphorothioate linkages.

Main Methods:

  • Development of synthetic DNA standards for primer editing analysis.
  • Utilizing next-generation sequencing for high-throughput enzymological measurements.
  • Comparative analysis of different polymerases and polymerase chain reaction (PCR) cycling conditions.
  • Investigating the effect of phosphorothioate linkages on primer editing.

Main Results:

  • Established primer editing standards enable accurate, sequencing-based quantification of primer editing extent.
  • Demonstrated that primer editing by proofreading polymerases is concentration-dependent.
  • Showed that primer editing can be modulated by phosphorothioate linkages.
  • Primer editing successfully rescued taxon dropout caused by primer mismatches in mock communities and microbiome samples.

Conclusions:

  • Synthetic DNA standards provide a robust platform for studying primer editing.
  • Primer editing is a tunable enzymatic activity of proofreading polymerases with practical applications in molecular diagnostics.
  • Primer editing can enhance the accuracy and reliability of amplicon sequencing, particularly in complex microbial communities.