Yonsei Med J.  2012 Jan;53(1):132-137. 10.3349/ymj.2012.53.1.132.

A Modified Extraction Method of Circulating Free DNA for Epidermal Growth Factor Receptor Mutation Analysis

• Yuan H
• Zhu ZZ
• Lu Y
• Liu F
• Zhang W
• Huang G
• Zhu G
• Jiang B

• KMID: 1779697
• DOI: http://doi.org/10.3349/ymj.2012.53.1.132

Abstract

PURPOSE
Circulating free DNA (cfDNA) in plasma is promising to be a surrogate for tumor tissue DNA. However, not all epidermal growth factor receptor (EGFR) mutations in tumor tissue DNA has been detected in matched cfDNA, at least partly due to inefficient cfDNA extraction method. The purpose of this study was to establish an efficient plasma cfDNA extraction protocol.
MATERIALS AND METHODS
The yield of plasma cfDNA extracted by our modified phenol-chloroform (MPC) method from non-small-cell lung cancer (NSCLC) patients was compared with that by QIAamp MinElute Virus Spin kit (Qiagen kit) as control, using the Wilcoxon rank-sum test. TaqMan quantitative polymerase chain reaction (qPCR) assays were used to quantify the plasma cfDNA extracted. Both Mutant-enriched PCR (ME-PCR) coupled sequencing and DxS EGFR mutation test kit were used to evaluate the impact of extraction method on EGFR mutation analysis.
RESULTS
MPC method extracted more plasma cfDNA than Qiagen kit method (p=0.011). The proportion of longer fragment (> or =202 bp) in cfDNA extracted by MPC method was significantly higher than by Qiagen kit method (p=0.002). In the sequencing maps of ME-PCR products, a higher mutant peak was observed on plasma cfDNA extracted by MPC method than by Qiagen kit method. In DxS EGFR mutation test kit results, plasma cfDNA extracted by MPC method contained more tumor-origin DNA than by Qiagen kit method.
CONCLUSION
An improved plasma cfDNA extraction method of MPC is provided, which will be beneficial for EGFR mutation analysis for patients with NSCLC.

Keyword

Circulating free DNA; DNA extraction; methodology; EGFR mutation; non-small-cell lung cancer

MeSH Terms

Base Sequence
Carcinoma, Non-Small-Cell Lung/*genetics
Chloroform
DNA Mutational Analysis/*methods
DNA, Neoplasm/*blood/*isolation & purification
Genetic Testing/methods
Humans
Lung Neoplasms/*genetics
Molecular Sequence Data
Phenol
Polymerase Chain Reaction/methods
Receptor, Epidermal Growth Factor/*genetics

Figure

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  Fig. 1 Comparison of cfDNA yield extracted by modified phenol-chloroform (MPC) method and QIAamp MinElute Virus Spin kit (Qiagen kit).

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  Fig. 2 Comparison of fragment distribution of plasma cfDNA extracted by modified phenol-chloroform (MPC) method and QIAamp MinElute Virus Spin kit (Qiagen kit).

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  Fig. 3 Sequencing maps of known EGFR deletion in exon 19 of plasma cfDNA. cfDNA was extracted by modified phenol-chloroform (MPC) method (A) and QIAamp MinElute Virus Spin kit (Qiagen kit) (B). In (A), the height of mutant peak is similar to that of wild-type peak, while the height of mutant peak in (B) is much lower than that of wild-type peak. EGFR, epidermal growth factor receptor.

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  Fig. 4 Amplification plot of cfDNA extracted by modified phenol-chloroform (MPC) method and QIAamp MinElute Virus Spin kit (Qiagen kit) in DxS EGFR mutation test kit. EGFR, epidermal growth factor receptor.

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