Received on April 1, 2008
Accepted on July 22, 2008

Molecular Diagnostics and Genetics

Genotyping -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, and Dipartimento di Medicina Sperimentale e Patologia, Universita' degli Studi "La Sapienza," Roma, Italy
⁵ Genomic Unit for the Diagnosis of Human Pathologies, San Raffaele Scientific Institute, Milano, Italy; Ricerca e Diagnostica San Raffaele, Milano, Italy, and Universita' Vita-Salute San Raffaele, Milano, Italy

^* To whom correspondence should be addressed. 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 -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 -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.