DNA polymerase characteristics influence noise levels in sequencing of short tandem repeats

Published

March 3, 2026

Author(s)

Tova Lindh, Maja Sidstedt, Kevin Kiesler, Peter Vallone, Johannes Hedman

Abstract

Polymerase chain reaction (PCR) applications including sequencing rely on accurate thermostable DNA polymerases. Polymerization errors may hinder the detection of low-level DNA variants such as mutations in clinical samples or DNA from minor contributors in crime scene traces. Short Tandem Repeat (STR) markers are particularly affected by artefacts. Apart from the regular random base substitutions, the repeated structure of STRs makes them prone to formation of stutter products. However, the mechanisms leading to stutter formation have not yet been fully elucidated. Here, we applied an STR assay based on Unique Molecular Identifiers (UMIs) to study the effects of DNA polymerases with different characteristics on the amplicon yield as well as the formation of PCR errors. The application of UMIs made it possible to study the impact on error formation of applying genomic DNA (mimicking the early PCR cycles) or amplicons (later cycles) as template. The levels of base substitutions were clearly connected to the fidelity of the DNA polymerases, which in turn was coupled with having an integrated 3'to 5' exonuclease domain. Stutter formation, on the other hand, was not as directly associated with fidelity, as two high-fidelity polymerases showed quite different levels of stutter. DNA binding domains generally improve processivity which could lower the incidence of stutter. However, this was not clear in the present study as a polymerase having a DNA binding domain gave the highest stutter levels. Overall, the degree of polymerase stuttering is likely due to several different DNA polymerase characteristics. Identifying a DNA polymerase that provides low levels of stutters and base substitutions may enable the detection of low-level variants such as DNA from minor contributors in mixed forensic traces.

Citation

BMC Genomics

Pub Type

Journals

Download Paper

https://doi.org/10.18434/mds2-4088

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Keywords

base substitutions, fidelity, in vitro polymerization, PCR, processivity, STR, stutter

Citation

Lindh, T. , Sidstedt, M. , Kiesler, K. , Vallone, P. and Hedman, J. (2026), DNA polymerase characteristics influence noise levels in sequencing of short tandem repeats, BMC Genomics, [online], https://doi.org/10.18434/mds2-4088, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=961080 (Accessed March 4, 2026)