Computational protocol: Comprehensive Genetic Screening of KCNQ4 in a Large Autosomal Dominant Nonsyndromic Hearing Loss Cohort: Genotype-Phenotype Correlations and a Founder Mutation

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Protocol publication

[…] All fourteen exons and flanking intronic sequences of the KCNQ4 gene were amplified by polymerase chain reaction PCR. Primers were designed to flank all of the exon-intron boundaries through use of the Primer3 web based server. Each genomic DNA sample (40 ng) was amplified using Multiplex PCR Assay Kit (Takara, Shiga, Japan) for 5 min at 95°C, followed by 40 three-step cycles of 94°C for 30 s, 60–67.6°C for 90 s, and 72°C for 90 s, with a final extension at 72°C for 10 min, ending with a holding period at 4°C in a Perkin-Elmer thermal cycler. The PCR products varied in size at about 100–400 bp, and they were treated with 0.1 ul exonuclease I (Amersham) and 1 ul shrimp alkaline phosphatase (Amersham) and by incubation at 37°C for 30 min, and inactivation at 80°C for 15 min. After the products were purified, we performed standard cycle sequencing reaction with ABI Big Dye terminators in an ABI 3100 autosequencer (Applied Biosystems).Computer analysis to predict the effect of missense variants on the protein function was performed with wANNOVAR (http://wannovar.usc.edu) including the functional prediction software listed below. PhyloP (http://hgdownload.cse.ucsc.edu/goldenPath/hg18/phyloP44way/), Sorting Intolerant from Tolerant (SIFT; http://sift.jcvi.org/), Polymorphism Phenotyping (PolyPhen2; http://genetics.bwh.harvard.edu/pph2/), LRT (http://www.genetics.wustl.edu/jflab/lrt_query.html), and MutationTaster (http://www.mutationtaster.org/). […]

Pipeline specifications

Software tools Primer3, wANNOVAR, PHAST, SIFT, PolyPhen, MutationTaster
Application qPCR
Organisms Homo sapiens