GJB2 p.G4D Genomic DNA Ref Std Deafness Res

Product Overview

This standard contains genomic DNA with a heterozygous p.G4D mutation, one of the most prevalent GJB2 variants associated with non-syndromic sensorineural deafness in multiple populations. With a precisely calibrated 35% mutant allele frequency, it mimics the genetic profile of clinical specimens, enabling accurate validation of diagnostic assays used in deafness research and clinical genetics laboratories. Manufactured under stringent quality controls, this standard addresses the critical need for reliable references in GJB2 testing, where accurate mutation detection directly impacts genetic counseling and clinical management .

Product Features

Clinically Actionable Mutation

The standard carries the p.G4D missense mutation (NM_004004.6:c.10G>A), which substitutes glycine with aspartic acid at codon 4 of the GJB2 protein (connexin 26). This mutation is classified as pathogenic by ACMG guidelines, associated with moderate-to-severe congenital hearing loss. The allele frequency is verified by ddPCR to ensure accuracy within ±5% of the nominal value .

Comprehensive Validation

Mutation status is confirmed through multiple orthogonal methods:

• Sanger sequencing with >99.9% base call accuracy

• Next-generation sequencing (>500x coverage across GJB2 exon 1)

• Digital droplet PCR quantification of mutant alleles

• Restriction fragment length polymorphism (RFLP) analysis

Each lot undergoes sequence validation across the entire 1,240 bp GJB2 coding region to exclude additional variants.

Research-Grade Quality

Purified to exceptional quality metrics:

• High molecular weight DNA (>25 kb)

• A260/A280 ratio of 1.85 ± 0.1

• <0.01% RNA contamination

• Absence of PCR inhibitors (verified by internal control amplification)

This ensures compatibility with sensitive research applications including allele-specific PCR, melting curve analysis, and high-throughput sequencing.

Usage

Assay Development

Use at a standard input of 20 ng DNA per reaction to optimize GJB2 p.G4D detection assays. Prepare serial dilutions (0.1-100 ng) to determine:

• Limit of detection (LOD) for mutation calling

• Assay linearity across clinically relevant allele frequencies

• Signal-to-noise ratio for variant discrimination

Research Applications

Incorporate into research studies to:

• Validate novel GJB2 sequencing panels

• Compare performance of different detection platforms

• Standardize mutation frequency quantification

• Assess bioinformatics pipeline accuracy for variant calling

Compatible with Illumina, Ion Torrent, and PacBio sequencing platforms.

Storage Protocol

Store unopened vials at -80°C for up to 24 months. After first use, aliquot into 5 μL volumes and store at -80°C to avoid freeze-thaw cycles. Thaw on ice for 10 minutes before use. Reconstituted DNA remains stable at 4°C for up to 48 hours when stored in a refrigerator.

FAQ

What is the clinical significance of p.G4D mutation?

The p.G4D mutation disrupts connexin 26 protein structure, impairing gap junction formation in the inner ear. This leads to abnormal potassium recycling in the cochlea, resulting in progressive sensorineural hearing loss that typically presents in early childhood. Detection enables early intervention and genetic counseling for affected families .

How does this standard support research?

It provides a consistent reference for comparing results across studies and laboratories, reducing inter-assay variability in GJB2 mutation detection. The well-characterized allele frequency allows accurate calibration of quantitative methods like ddPCR and NGS .

Is it suitable for validation of multiplex panels?

Yes, the standard is validated for use in multi-gene deafness panels containing GJB2, GJB6, SLC26A4, and other deafness-associated genes. It includes a detailed genotype report to confirm absence of variants in these additional genes .

What controls are provided for specificity testing?

Each standard includes a wild-type GJB2 control from the same genetic background, enabling researchers to verify assay specificity and establish background noise levels for mutation calling.