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

BMC Clinical Pathology

Darryl Shibata²  Myron F Goodman¹  Nancy Quach² 

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

[2]Department of Pathology, University of Southern California Keck School of Medicine, Los Angeles, CA, USA

Others  :  1086460
DOI  :  10.1186/1472-6890-4-1

 received in 2003-12-02, accepted in 2004-02-12,  发布年份 2004

【 摘 要 】

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.

【 授权许可】

   
2004 Quach et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.

【 图 表 】

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