Journal Article

High dynamic range capillary electrophoresis method for sensitive detection of low-frequency driver mutations

Maeda, Chiho, Chiba, Shin-ichi, Mizukami, Yusuke, Takahashi, Kenji, Tanino, Mishie, Hagiwara, Yoshihiko, Tamamura, Nobue, Koyama, Kazuya, Sato, Hiroki, Anazawa, Takashi, Kawabata, Hidemasa, Ono, Yusuke, Inaba, Ryoji, Okada, Tetsuhiro, Kato, Hirokazu, Ohtaki, Yu, Mori, Miyuki, Taniue, Kenzui

2025

Overview

Cancer genomics aims to personalize treatments by identifying genetic abnormalities in cancer cells. However, current analytical techniques face limitations in simplicity and cost-effectiveness. To address these issues, we developed an enhanced capillary gel electrophoresis (CE) sequencer using a fluorescence-acquisition technique called “HiDy” (High Dynamic range) (HiDy-CE). The HiDy-CE reduces the hardware binning region size and increases the number of regions on a charge-coupled device image sensor, expanding the dynamic range and reducing saturation risk. By applying the multi-base primer extension method to the HiDy-CE with control DNA containing known mutations, we detected variant allele frequencies (VAFs) as low as 0.5% for major KRAS hotspot mutation at codon 12 and 13. With 10 ng of DNA from small tissues obtained via fine-needle biopsy from patients with suspected pancreaticoduodenal tumors, HiDy-CE produced equivalent VAFs in KRAS compared with targeted amplicon sequencing. This demonstrated the world’s first capability of detecting mutations below 1% on CE using pathological specimens, leveraging its wide dynamic range. With only 2 ng of input DNA, the HiDy-CE provided results highly concordant with digital PCR with minimal non-specific noise. These findings underscore the HiDy-CE’s potential for sensitive detection of oncogenes such as KRAS , facilitating pre-testing before comprehensive genome profiling.

• Publisher: Nature Publishing Group UK,Nature Portfolio
• Subject: 692/4020 / 692/53 / 692/53/2421 / Capillary gel electrophoresis / DNA Mutational Analysis - methods / Dynamic range / Electrophoresis, Capillary - methods / Fragment analysis / Gene Frequency / HiDy / Humanities and Social Sciences / Humans / KRAS / Low-frequency mutation / multidisciplinary / Mutation / Pancreatic Neoplasms - genetics / Proto-Oncogene Proteins p21(ras) - genetics / Science / Science (multidisciplinary)