HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: clinchem.2008.107870v1 54/10/1657    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Battistella, S. Articles by Cremonesi, L. Search for Related Content PubMed PubMed Citation Articles by Battistella, S. Articles by Cremonesi, L.

Genotyping β-Globin Gene Mutations on Copolymer-Coated Glass Slides with the Ligation Detection Reaction

¹ Genomic Unit for the Diagnosis of Human Pathologies, San Raffaele Scientific Institute, Milano, Italy; ² Istituto di Chimica del Riconoscimento Molecolare (ICRM), C.N.R., Milano, Italy; ³ Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, PA; ⁴ Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA; ⁵ Dipartimento di Medicina Sperimentale e Patologia, Universita’ degli Studi "La Sapienza," Roma, Italy; ⁶ Ricerca e Diagnostica San Raffaele, Milano, Italy; ⁷ Universita’ Vita-Salute San Raffaele, Milano, Italy.

^aAddress correspondence to this author at: Genomic Unit for the Diagnosis of Human Pathologies, DIBIT2, San Raffaele Scientific Institute, Via Olgettina 60 Milan, 20132 Milan, Italy. Fax 0039 02 26434351; e-mail cremonesi.laura{at}hsr.it.

Background: Methods are needed to analyze small amounts of samples for variation in disease-causing genes. One means is to couple the sensitivity and multiplexing capability of the ligation detection reaction (LDR) with the use of simple glass slides specifically functionalized with a novel polymer coating to enhance sensitivity.

Methods: We developed an array-based genotyping assay based on glass slides coated with copolymer (N,N-dimethylacrylamide, N,N-acryloyloxysuccinimide, and 3-(trimethoxysilyl)propyl methacrylate). The assay consists of an LDR with genomic DNA followed by a universal PCR (U-PCR) of genomic DNA–templated LDR product. The LDR occurs in the presence of 3 primers for each sequence variant under investigation: 2 distinguishing primers (allele specific and perfectly complementary to wild-type and mutant alleles) and 1 common locus-specific primer. The 2 allele-specific primers have different capture sequences for binding different complementary probes on a tag array. The LDR product templated from genomic DNA is made fluorescent during the U-PCR via incorporation of a Cy5-labeled universal primer into all LDR products; detection occurs on the coated glass slides.

Results: The assay was designed to detect 7 prevalent mutations in the β-globin gene (HBB, hemoglobin, beta) in a multiplex format, and signals for the different alleles are detected by their fluorescence. The assay was applied to 40 genomic DNA samples from both control individuals and patients with known β-thalassemia mutations. Results show good correspondence between the patients’ genotypes as assessed by DNA sequence analysis and those generated from the LDR assays.

Conclusions: The developed technology allows accurate identification of sequence variants in a simple, cost-effective way and offers good flexibility for scaling to other applications with different numbers of single-nucleotide polymorphisms or mutations to be detected.