title: Integrated PCR amplification and detection processes on a Lab-on-Chip platform: a new advanced solution for molecular diagnostics.
abstract: BACKGROUND
  Several microdevices have been developed to perform only a single step of a genotyping process, such as PCR or detection by probe hybridization. Here, we describe a Lab-on-Chip (LoC) platform integrating a PCR amplification microreactor with a customable microarray for the detection of sequence variations on human genomic DNA.
  
  
  METHODS
  Preliminary work was focused on developing the single analytical steps including PCR and labeling strategies of the amplified product by conventional reference systems. The optimized protocols included a 1:4 forward:reverse primer ratio for asymmetric PCR, and Cy5-dCTP multiple incorporation for the generation of a labeled PCR product to be hybridized to complementary probes bound to the chip surface.
  
  
  RESULTS
  Final conditions were applied to the fully integrated LoC platform for the detection of the IVSI-110 G > A mutation in the human beta-globin (HBB) gene associated with beta-thalassemia, used as a model of genetic application, allowing for correct genotyping of 25 samples that were heterozygous, homozygous or wild-type for this mutation.
  
  
  CONCLUSIONS
  The overall results show that the present platform is very promising for rapid identification of DNA sequence variations in an integrated, cost effective and convenient silicon chip format.
authors.affiliation: Genomic Unit for the Diagnosis of Human Pathologies, Center for Genomics, Bioinformatics and Biostatistics, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Foglieni
authors.forename: Angela
authors.initials: A
authors.lastname: Brisci
authors.forename: Floriana
authors.initials: F
authors.lastname: San Biagio
authors.forename: Patrizia
authors.initials: P
authors.lastname: Di Pietro
authors.forename: Salvatore
authors.initials: S
authors.lastname: Petralia
authors.forename: Sabrina
authors.initials: S
authors.lastname: Conoci
authors.forename: Maurizio
authors.initials: M
authors.lastname: Ferrari
authors.forename: Laura
authors.initials: L
authors.lastname: Cremonesi
mesh_terms: D002232:Carbocyanines / Q000737:chemistry / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000737:chemistry / Q000378:metabolism; D014644:Genetic Variation; D015894:Genome, Human; D005838:Genotype; D006801:Humans; D056656:Lab-On-A-Chip Devices; D025202:Molecular Diagnostic Techniques / Q000295:instrumentation* / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D013789:Thalassemia / Q000235:genetics; D055544:beta-Globins / Q000235:genetics
chemical_list: D002232:Carbocyanines; C544355:Cy5-dCTP; D003845:Deoxycytosine Nucleotides; D055544:beta-Globins