Double Mutation β-Thalassemia Ref Std Research

Product Overview

The Double Mutation β-Thalassemia Ref Std Research is a specialized reference material designed for validating research workflows investigating complex β-thalassemia genotypes. This standard contains genomic DNA with two clinically significant β-globin gene mutations: IVS-II-654 (C>T) and CD41-42 (-TCTT), present in compound heterozygous state. These mutations represent one of the most prevalent mutation combinations associated with β-thalassemia intermedia in Southeast Asian populations, where this genotype accounts for approximately 35% of clinically significant β-thalassemia cases. Engineered to mimic the genetic complexity of clinical specimens, this standard enables accurate validation of multi-mutation detection assays used in β-thalassemia research and therapeutic development .

Product Features

Complex Genotype Representation

Carries two pathogenic β-thalassemia mutations in trans configuration:

• IVS-II-654 (HBB:c.315+654C>T): A splice site mutation in intron 2

• CD41-42 (-TCTT) (HBB:c.126_129delTCTT): A 4-base pair deletion in exon 2

Each mutation is quantified at ~50% allele frequency by ddPCR, reflecting the compound heterozygous state. Mutation status is verified by haplotype analysis to confirm trans configuration .

Multi-Platform Validation

Mutation detection is confirmed across multiple research platforms:

• Long-read NGS (>100x coverage across HBB gene cluster)

• Sanger sequencing of β-globin gene (exon 1-3 and flanking regions)

• Multiplex ARMS-PCR with allele-specific primers

• Restriction fragment length polymorphism (RFLP) analysis

Whole-genome sequencing confirms absence of additional hemoglobin variants.

Research-Ready Format

Purified to support advanced research applications:

• High molecular weight DNA (>40 kb) suitable for long-read sequencing

• <0.001% microbial contamination (verified by 16S rRNA PCR)

• Preserved epigenetic modifications in the β-globin locus control region

• Compatible with CRISPR editing efficiency assessment workflows

Usage

Assay Development

Use at 100 ng input for optimization of multi-mutation detection assays:

• Design and validate multiplex PCR panels

• Optimize NGS library preparation from low-quality samples

• Establish bioinformatics pipelines for complex genotype calling

• Develop allele-specific quantification methods

Therapeutic Research Applications

Incorporate into gene therapy research to:

• Validate gene editing efficiency for both mutations

• Assess allele-specific expression changes

• Monitor off-target effects in β-globin locus editing

• Standardize functional assays of hemoglobin production

Storage Requirements

Store at -80°C in original packaging for up to 48 months. After first use, aliquot into single-use volumes (50 μL) and store at -80°C. Avoid more than 2 freeze-thaw cycles. Thaw on ice for 20 minutes before use and mix gently by pipetting.

FAQ

Why is this double mutation combination clinically important?

The IVS-II-654/CD41-42 genotype typically results in β-thalassemia intermedia, characterized by moderate anemia requiring occasional transfusions. This genotype exhibits variable clinical severity, making it an important model for studying modifier genes and developing targeted therapies .

How does this standard support research reproducibility?

It provides a consistent reference for comparing results across studies investigating complex β-thalassemia genotypes. The defined mutation frequencies enable accurate calibration of quantitative assays, reducing inter-laboratory variability in mutation detection and expression analysis .

Can it be used for validation of prenatal diagnosis methods?

Yes, the standard is suitable for validating non-invasive prenatal testing (NIPT) methods for β-thalassemia, including cell-free DNA analysis. It enables assessment of assay sensitivity for detecting both mutations in mixed DNA backgrounds .

What additional characterization data is provided?

Each lot includes comprehensive haplotype analysis of the β-globin gene cluster (HBB, HBD, HBG1, HBG2), methylation profiling of the locus control region, and expression data from erythroid progenitor cell culture models.