Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Dramatic reduction of sequence artefacts from DNA isolated from formalin-fixed cancer biopsies by treatment with uracil- DNA glycosylase.

Author(s): Do H, Dobrovic A

Publication: Oncotarget, 2012, Vol. 3, Page 546-58

PubMed ID: 22643842 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of template amount and UDG treatment on identification of sequence mutations using DNA from FFPE lung carcinoma specimens. DNA was extracted from 73 FFPE lung carcinoma specimens using the DNeasy kit and was stored for 7-17 years.

   Summary of Findings:

   15 of the 73 specimens were identified to have sequence variants in RAC-alpha serine/threonine-protein kinase (AKT1) by HRM analysis, but none of the variants were the known E17K variant. All of the identified variants were single base substitutions, and 16 of 17 identified mutations in AKT1 were C:G>T:A. When the input was increased from 5 ng to 25 ng, 4 of 5 specimens were found to be wild-type. Adding UDG (0.1-1 units/reaction) also reduced mutation identification and did not affect cycle threshold value. In 3 specimens that had AKT1 mutations identified in 57% (34 of 60), 40% (24 of 60) and 33% (20 of 60) of replicates by low copy number (LCN) HRM analysis, treatment with UDG decreased the mutation frequency to 8% (5 of 60), 17% (10 of 57) and 5% (3 of 57), respectively. UDG treatment resulted in a similar reduction in mutations identified by sequencing from 12 in 10 reactions to 7 in 32 reactions with all 7 remaining mutations consistent with deamination of 5-methylcytosine to thymine. Further, UDG treatment reduced the number of mutations identified in proto-oncogene B-Raf (BRAF) exon 15 (15/46 to 0/48) and epidermal growth factor receptor (EGFR) exon 19 (16/29 to 1/28). However, UDG treatment did not affect detection of true mutations in KRAS and EGFR.

   Biospecimens

   • Tissue - Lung

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	SNP assay
   DNA	DNA sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   SNP assay Specific	Targeted nucleic acid	AKT1 EGFR BRAF
   SNP assay Specific	Technology platform	Sequencing HRM
   SNP assay Specific	Template/input amount	5 ng 25 ng
   SNP assay Specific	Template modification	No UDG added 0.1 units UDG/reaction 0.25 units UDG/reaction 0.5 units UDG/reaction 1.0 units UDG/reaction

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