Novel multiplex primer extension and denaturing high-performance liquid chromatography for genotyping of the deafness gene mutations
10.3969/j.issn.1672-7347.2011.11.008
- VernacularTitle:一种新的高效快速检测遗传性耳聋的方法
- Author:
Meichao MEN
;
Jinjie XUE
;
Lu JIANG
;
Honghan WANG
;
Qian PAN
;
Yong FENG
- Publication Type:Journal Article
- Keywords:
genetic deafness;
denaturing high-performance liquid chromatography;
mutation;
primer extension
- From:
Journal of Central South University(Medical Sciences)
2011;36(11):1079-1084
- CountryChina
- Language:Chinese
-
Abstract:
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.
