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Updated: May 26, 2026
Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry
Published on: June 21, 2018
Novel multiplex primer extension and denaturing high-performance liquid chromatography for genotyping of the
Meichao Men1, Jinjie Xue, Lu Jiang
1Department of Otolaryngology-Head and Neck Surgery, Xiangya Hospital, Central South University, Changsha 410008, China.
Objective:
To find a rapid and accurate genotyping method for specific non-syndromic hearing loss (NSHL)-causing gene mutations for disease diagnosis in different ethnic populations.
Methods:
We performed a novel multiplex primer extension (PE) reaction in combination with denaturing high-performance liquid chromatography (DHPLC) to simultaneously detect and genotype the 6 most common mutations in 180 patients with NSHL (GJB2-235delC, GJB2-299delAT, PDS-A2168G, PDS IVS7-2A>G, mtDNA-A1555G, and mtDNA-C1494T) in Chinese population. This method involved the amplification of the target sequence, followed by a purification step, a multiplex PE reaction, and DHPLC analysis performed on the Transgenomic Wave DNA fragment analysis system under fully-denaturing conditions.
Results:
In a blind analysis, this technique successfully and accurately genotyped 100% of the samples simultaneously characterized by direct sequencing.
Conclusion:
Combination of PE and DHPLC is simple, rapid, accurate, and cost-effective for genotyping common disease-causing mutations, including substitutions, insertions, and deletions in NSHL, and may be successfully used in other genetic diseases.
