Identifiable universal fluorescent multiplex PCR equipped with capillary electrophoresis for genotyping of exons 1 to 5 in human red and green pigment genes

Congenital red and green color blindness is the most X-linked recessive disorder in humans caused by deletions or gross structural rearrangements of the visual pigment gene array that lead to altered the functions of visual pigments in their retina differ from normal. The incidence is about 7-10% in male and close association of Xlinked recessive disorders (such as: hemophilia A, hemophilia B, duchenne muscular dystrophy). However, the traditional genetic analysis methods are time-consuming and low-efficiencies. Therefore, the purpose of the study is to develop a rapid method for genotyping of red and green pigment genes. We describe herein the first method for simultaneous evaluation of ten exons in the red and green pigment genes for genetic analysis. A forward specific primers with identifiable universal fluorescent multiplex PCR (FSIUFM-PCR) method utilized one universal primer (containing two universal non-human sequences) and forward specific primers in the multiplex PCR reaction system for simultaneously fluorescent labeling of eleven gene fragments (ten exons in red and green pigment genes and one internal standard). All the PCR products were analyzed on capillary electrophoresis with short-end injection, which had the advantage of high resolution and rapid separation. Of all 80 detected individuals, 7 subjects with color vision deficiencies (including 3 subjects only had red exons 1-5, 4 subjects had a specific red-green or green-red hybrid gene and 73 subjects with normal color vision). All genotyping results showed good agreement with DNA sequencing data. This method provided a better potential technique for genotyping and identifying of red and green pigment genes. In addition, FSIUFM-PCR method will be useful in many fields, such as diagnosis of diseases, analysis of polymorphisms and quantitative assay.

Automated summary

A rapid method for genotyping of red and green pigment genes is developed in this study. Using fluorescence labeling and capillary electrophoresis analysis, this method can accurately detect individuals with color vision deficiencies and shows good agreement with DNA sequencing results.