Clinical Chemistry Link to Randox Laboratories Web Site
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 48: 952-955, 2002;
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, W.
Right arrow Articles by Chong, S. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, W.
Right arrow Articles by Chong, S. S.
Related Collections
Right arrow Molecular Diagnostics and Genetics
Right arrow Laboratory Management
(Clinical Chemistry. 2002;48:952-955.)
© 2002 American Association for Clinical Chemistry, Inc.

Technical Briefs

"Reconstituted" {alpha}-Thalassemia Genomic Samples as Positive Controls for the Molecular Diagnostic Laboratory

Wen Wang1, Arnold S-C. Tan1 and Samuel S. Chong1,2,3a

1 Departments of Pediatrics and Obstetrics and Gynecology, National University of Singapore, Singapore 119074, Singapore

2 Molecular Diagnosis Center, Department of Laboratory Medicine, National University Hospital, Singapore 119074, Singapore

3 Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, MD 21205

aaddress correspondence to this author at: Department of Pediatrics, National University of Singapore, Level 4, Main Building, National University Hospital, 5 Lower Kent Ridge Rd., Singapore 119074, Singapore; fax 65-6779-7486, e-mail paecs@nus.edu.sg

We and others recently described strategies for multiplex-PCR analysis of deletional determinants of {alpha}-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 {alpha}-thalassemia genotype, derived from existing patient DNA samples. Briefly, genomic DNA of known {alpha}-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 {alpha}-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 . . . [Full Text of this Article]


Acknowledgments


References






HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 2002 by the American Association for Clinical Chemistry.