In vitro mutation artifacts after formalin fixation and error prone translesion synthesis during PCRReport as inadecuate




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BMC Clinical Pathology

, 4:1

First Online: 12 February 2004Received: 02 December 2003Accepted: 12 February 2004

Abstract

BackgroundClinical 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.

MethodsTo 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.

ResultsSignificantly 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.

ConclusionsFormalin 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.

Electronic supplementary materialThe online version of this article doi:10.1186-1472-6890-4-1 contains supplementary material, which is available to authorized users.

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Author: Nancy Quach - Myron F Goodman - Darryl Shibata

Source: https://link.springer.com/



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