“Reconstituted” α-Thalassemia Genomic Samples as Positive Controls for the Molecular Diagnostic Laboratory

Abstract

We and others recently described strategies for multiplex-PCR analysis of deletional determinants of α-thalassemia (1)(2)(3), culminating in the development of a single-tube assay for simultaneous screening of seven common deletions (4). Since then, several molecular diagnostic laboratories wanting to set up the test have requested DNA samples carrying these deletions for use as validation and positive controls. These requests have led to a critical shortage of our limited stocks of genomic samples, especially those with the rarer deletions, something that has caused our inability to fulfill all requests. The ideal solution to limited genomic DNA is to establish immortal lymphoblastoid cell lines from peripheral-blood leukocytes of patients by Epstein–Barr virus transformation (5)(6). To do so, however, requires that patients be contacted again to provide renewed consent and a fresh aliquot of blood for transformation, something that may be inconvenient or impractical for third-party referral laboratories to implement. We have devised an alternative strategy for creating a renewable resource of positive control samples of known α-thalassemia genotype, derived from existing patient DNA samples. Briefly, genomic DNA of known α-thalassemia genotype was initially used as a template to amplify each deletion junction fragment individually by PCR. The relevant primer pairs and thermo-cycling conditions were as described previously (4). Junction fragments of seven α-thalassemia deletions were PCR-amplified separately from patient DNA, gel-purified with the GFXTM reagent set (Amersham Pharmacia Biotech), and ligated to a pBluescript (Stratagene) T-vector (Fig. 1A⇓ ). Each 5-μL ligation reaction contained 15 ng of T-vector and an amount of …