PCR/ Sequencing for EGFR mutation analysis

The diagram below summarises the PCR/sequencing for the EGFR mutation analysis process.

Mouse-over to enlarge

Several sequencing-based technologies can be used for EGFR mutation testing, including

Best practice for PCR reactions

•    Include positive and negative controls
•    Two independent PCR reactions should be carried out if possible
•    Keep the risk of contamination low

Best practice for sequencing  (Eberhard 2008)

•    Include positive and negative controls
•    Forward and reverse sequencing reactions should be carried out
•    Reduce the risk of contamination by using separate pre- and post-PCR areas
•    Capillary sequencing should be carried out using robust standard operating procedures
•    When reading sequencing traces (either by eye or using sequencing software), the results of sequencing analysis by one operator should always be checked by another operator
•    When using sequencing analysis software, the settings should be set lower than those used for the detection of genetic polymorphisms in germline DNA
•    If novel mutations are identified, they must be verified

A downloadable document providing best practice information for EGFR PCR/Sequencing is available here

The diagram below shows DNA sequence analysis showing two different EGFR mutations visualised using Mutation Surveyor Software (SoftGenetics)