NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis
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In vitro mutation artifacts after formalin fixation and error prone translesion synthesis during PCR.

Author(s): Quach N, Goodman MF, Shibata D

Publication: BMC Clin Pathol, 2004, Vol. 4, Page 1

PubMed ID: 15028125 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to determine the effects of formalin fixation and fixation duration on PCR yield, maximum amplicon size and the number of mutations identified.

Conclusion of Paper

Increasing fixation duration led to decreasing PCR yield and decreased success in amplifying longer fragments. Fixed specimens had 3-4 fold more mutations than fresh specimens, but the number of mutations identified was not affected by fixation duration or fragment size. 92% of the mutations identified in fixed specimens were transition mutations, and 6.6% were small deletions or additions. Mutations were more likely to occur at AT pairs than GC pairs. The mutations identified were scattered evenly throughout the gene fragments. The authors report that using AmpliTaq gold rather than AmpliTaq increased PCR yields but did not affect mutation rates.

Studies

  1. Study Purpose

    The purpose of this study was to determine the effects of formalin fixation and fixation duration on PCR yield, maximum amplicon size and the number of mutations identified. Normal adjacent colon from a patient with adenocarcinoma was analyzed fresh or fixed in formalin for 1-7 days and paraffin embedded. Sections were deparaffinized with ClearRight 3, digested for 4 h in proteinase K at 56 degrees C, and boiled for 5 min in digestion buffer. DNA from fresh tissue was extracted using the DNeasy kit. DNA was cloned into the TOPO vector before sequencing.

    Summary of Findings:

    While all three products could be amplified in fresh specimens and those fixed for 1 day, only the 420 bp fragment could be amplified in specimens fixed for 3 days, and no products were amplified in specimens fixed for 7 days. Further, the yield of the 420 bp product was reduced in specimens fixed for 3 days compared to those fixed for 1 day. Fixed specimens had 3-4 fold more mutations than fresh specimens (P<0.05), but the number of mutations identified was not affected by fixation duration or fragment size. 92% of the mutations identified in fixed specimens were transition mutations (A-G or C-T), and 6.6% were small deletions or additions of short repeats. Further, mutations were more likely to occur at AT pairs than GC pairs (2.9:1, versus 1.2:1, p=0.034). The mutations identified were scattered evenly throughout the gene fragments. The authors report that using AmpliTaq gold rather than AmpliTaq increased PCR yields but did not affect mutation rates.

    Biospecimens
    Preservative Types
    • Formalin
    • None (Fresh)
    Diagnoses:
    • Normal
    • Neoplastic - Normal Adjacent
    Platform:
    AnalyteTechnology Platform
    DNA PCR
    DNA DNA sequencing
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
    None (fresh)
    Biospecimen Preservation Time in fixative 1 day
    3 days
    7 days
    PCR Specific Targeted nucleic acid Mutation cluster region of the APC gene
    PCR Specific Length of gene fragment 420 bp
    589 bp
    1007 bp
    PCR Specific Nucleic acid amplification AmpliTaq
    AmpliTaq gold
    View more

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