title: Genotyping beta-globin gene mutations on copolymer-coated glass slides with the ligation detection reaction.
abstract: 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 beta-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 beta-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.
authors.affiliation: Genomic Unit for the Diagnosis of Human Pathologies, San Raffaele Scientific Institute, Milano, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Battistella
authors.forename: Francesco
authors.initials: F
authors.lastname: Damin
authors.forename: Marcella
authors.initials: M
authors.lastname: Chiari
authors.forename: Kathleen
authors.initials: K
authors.lastname: Delgrosso
authors.forename: Saul
authors.initials: S
authors.lastname: Surrey
authors.forename: Paolo
authors.initials: P
authors.lastname: Fortina
authors.forename: Maurizio
authors.initials: M
authors.lastname: Ferrari
authors.forename: Laura
authors.initials: L
authors.lastname: Cremonesi
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D005838:Genotype; D005914:Globins / Q000235:genetics*; D006801:Humans; D009154:Mutation; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis
chemical_list: D017931:DNA Primers; D005914:Globins