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Open Access Research article

In vitro mutation artifacts after formalin fixation and error prone translesion synthesis during PCR

Nancy Quach1, Myron F Goodman2 and Darryl Shibata1*

Author Affiliations

1 Department of Pathology, University of Southern California Keck School of Medicine, Los Angeles, CA, USA

2 Hedco Molecular Biology Laboratories, Department of Biological Sciences and Chemistry, University of Southern California, Los Angeles, CA, USA

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BMC Clinical Pathology 2004, 4:1  doi:10.1186/1472-6890-4-1

Published: 12 February 2004

Abstract

Background

Clinical specimens are routinely fixed in 10% buffered formalin and paraffin embedded. Although DNA is commonly extracted from fixed tissues and amplified by PCR, the effects of formalin fixation are relatively unknown. Formalin fixation is known to impair PCR, presumably through damage that blocks polymerase elongation, but an insidious possibility is error prone translesion synthesis across sites of damage, producing in vitro artifactual mutations during PCR.

Methods

To better understand the consequences of fixation, DNA specimens extracted from fresh or fixed tissues were amplified with Taq DNA polymerase, and their PCR products were cloned and sequenced.

Results

Significantly more (3- to 4-fold) mutations were observed with fixed DNA specimens. The majority of mutations were transitions, predominantly at A:T base pairs, randomly distributed along the template.

Conclusions

Formalin fixation appears to cause random base damage, which can be bridged during PCR by Taq DNA polymerase through error prone translesion synthesis. Fixed DNA is a damaged but "readable" template.