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LIPOChip
 

LIPOChip

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Es el primer DNAchip para el diagnóstico de la Hipercolesterolemia Familiar, con marcado CE.

Es el primer DNAchip para el diagnóstico de la Hipercolesterolemia Familiar, con marcado CE.

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    LIPOChip LIPOChip Presentation Transcript

    •  
    • The first DNA chip with CE MARK DETECTION OF THE MOST FREQUENT FH MUTATIONS AND COPY NUMBER CHANGES OF THE LDLR GENE
    • What is LIPOchip?
      • Diagnosis of Familial Hypercholesterolemia
        • Detection of the most frequent mutations
          • LDLR gene
          • APOB gene
          • PCSK9 gene
          • Detection of Copy Number Variations
    • Biochip Chip/Array : DNA fragments (15-60bp) printed (spotted) on a solid support (glass) in an ordered way
    • Size of one spot : 50-100 μm 60-75mm Biochip
    • MOLECULAR BASIS OF HYBRIDIZATION
      • 1. Coated glass DNA chip : Contains specific oligos complementary to the mutations of interest
        • Chip size : 25 X 40 mm
        • Arrangement: 32 subarrays (20x20)
        • Number of replicates for each oligo: 10 spots
        • 2. Number of mutations:
        • 242 LDLR mutations
        • 3 APOB mutations
        • 6 PCSK9 mutations
        • 3. Included controls
        • External controls (synthetic oligos)
        • Internal controls (for checking copy number changes):
          • From chromosome 21
          • From chromosome X
    • MOLECULAR BASIS OF HYBRIDIZATION A T C G T A G C AMPLIFICATION (PCR) ( region of interest) FRAGMENTATION DNAse: random cuts Alkaline Phosphatase : 3´ ends free 5´ 5´ 3´ 3´ LABELLING Biotin : Indirect labelling Patient DNA (COMPLEMENTARITY) + HYBRIDIZATION DNAchip With oligos specific to the mutations
    • N M Homozygous donor Heterozygous donor Technical base N N N M M M N N N
    • Experimental procedure DNA-chip design & printing Hybridization optimization SPECIFICATIONS Mutations/SNPs to be analyzed Image Capture and Software development GENOTYPE Oligonucleotide design Multiplex PCR design
    • Oligonucleotide design
      • Length of the oligonucleotides range from 19-27 nucleotides
      • Target nucleotide always in the central position of the oligonucleotides in order to maximize the specificity of the hybridization
      • Probes are specific for the normal or the mutant allele
      Allele-Specific Oligonucleotides: TTTCTAGCAGG G GGAGGAGTTTG TTTCTAGCAGG C GGAGGAGTTTG 11nt wt mut 11nt
    • Oligonucleotide design
      • 4 oligos for each mutation which are generally
      • - 2 PerfectMatch PM (sense and antisense)
      • - 2 MisMatch MM (sense and antisense)
      • Each oligo tested and validated with at least one patient
      • - Specificity
      • - Sensibility
    • Multiplex PCR Design M1 M2 M3 Exon 1 M4 M5 Exon 6
    • Multiplex PCR Design
      • Individual amplification
    • Multiplex PCR Design
      • Multiplex amplification
      A B C
    • Genotypes computing
      • Based on intensity values of normal and mutated oligos :
    • Detection of Copy Number Changes
      • Specific controls included in the chip and in each PCR group :
        • Normalization : Chromosome 21
        • Copy number change detection : Chromosome X
        • In each batch of hybridization, male and female controls are processed
        • Based on ratio of intensities of hybridization
    • DAY1 DAY 2 OVERLAPPING PROCESSES 56 ul WORKFLOW 1 Amplification 7.5µl DNA (20ng/µl) 2 Fragmentation 3 Labelling 4 Hybridization 5 Results analysis 24 ul PCR mixes 1, 2 y 3 and 4 DNAse + Alkaline Phosphatase TdT + Biotin-ddUTP 2 hours 45 minutes 60 minutes 4 hours and 30 minutes
    • ESSENTIAL EQUIPMENT REQUIRED FOR PROCESSING 1. Thermocycler: Applied Biosystem 9700
      • Patient DNA amplification (± 2h)
      • Fragmentation and Labelling (±1h45)
    • ESSENTIAL EQUIPMENT REQUIRED FOR PROCESSING 2.- Hybridization station: Tecan HS 4800™ Pro Hybridization Stations
      • Hybridization of the amplified, fragmented and labelled DNAs
      • An “easy to use” software controlls all the process.
      • 12 samples can be processed at the same time.
    • 3. Scanner: Innoscan 710A (Innopsys)
      • Hybridized slides scanning (1 minute per slide)
      • Macros and software installed on the same computer for data analysis
      ESSENTIAL EQUIPMENT REQUIRED FOR PROCESSING
    • ESSENTIAL EQUIPMENT REQUIRED FOR PROCESSING 4. LIPOchip software: result analysis and report