LIPOChip

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

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LIPOChip

  1. 2. The first DNA chip with CE MARK DETECTION OF THE MOST FREQUENT FH MUTATIONS AND COPY NUMBER CHANGES OF THE LDLR GENE
  2. 3. What is LIPOchip? <ul><li>Diagnosis of Familial Hypercholesterolemia </li></ul><ul><ul><li>Detection of the most frequent mutations </li></ul></ul><ul><ul><ul><li>LDLR gene </li></ul></ul></ul><ul><ul><ul><li>APOB gene </li></ul></ul></ul><ul><ul><ul><li>PCSK9 gene </li></ul></ul></ul><ul><ul><ul><li>Detection of Copy Number Variations </li></ul></ul></ul>
  3. 4. Biochip Chip/Array : DNA fragments (15-60bp) printed (spotted) on a solid support (glass) in an ordered way
  4. 5. Size of one spot : 50-100 μm 60-75mm Biochip
  5. 6. MOLECULAR BASIS OF HYBRIDIZATION <ul><li>1. Coated glass DNA chip : Contains specific oligos complementary to the mutations of interest </li></ul><ul><ul><li>Chip size : 25 X 40 mm </li></ul></ul><ul><ul><li>Arrangement: 32 subarrays (20x20) </li></ul></ul><ul><ul><li>Number of replicates for each oligo: 10 spots </li></ul></ul><ul><ul><li>2. Number of mutations: </li></ul></ul><ul><ul><li>242 LDLR mutations </li></ul></ul><ul><ul><li>3 APOB mutations </li></ul></ul><ul><ul><li>6 PCSK9 mutations </li></ul></ul><ul><ul><li>3. Included controls </li></ul></ul><ul><ul><li>External controls (synthetic oligos) </li></ul></ul><ul><ul><li>Internal controls (for checking copy number changes): </li></ul></ul><ul><ul><ul><li>From chromosome 21 </li></ul></ul></ul><ul><ul><ul><li>From chromosome X </li></ul></ul></ul>
  6. 7. 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
  7. 8. N M Homozygous donor Heterozygous donor Technical base N N N M M M N N N
  8. 9. 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
  9. 10. Oligonucleotide design <ul><li>Length of the oligonucleotides range from 19-27 nucleotides </li></ul><ul><li>Target nucleotide always in the central position of the oligonucleotides in order to maximize the specificity of the hybridization </li></ul><ul><li>Probes are specific for the normal or the mutant allele </li></ul>Allele-Specific Oligonucleotides: TTTCTAGCAGG G GGAGGAGTTTG TTTCTAGCAGG C GGAGGAGTTTG 11nt wt mut 11nt
  10. 11. Oligonucleotide design <ul><li>4 oligos for each mutation which are generally </li></ul><ul><li>- 2 PerfectMatch PM (sense and antisense) </li></ul><ul><li>- 2 MisMatch MM (sense and antisense) </li></ul><ul><li>Each oligo tested and validated with at least one patient </li></ul><ul><li>- Specificity </li></ul><ul><li>- Sensibility </li></ul>
  11. 12. Multiplex PCR Design M1 M2 M3 Exon 1 M4 M5 Exon 6
  12. 13. Multiplex PCR Design <ul><li>Individual amplification </li></ul>
  13. 14. Multiplex PCR Design <ul><li>Multiplex amplification </li></ul>A B C
  14. 15. Genotypes computing <ul><li>Based on intensity values of normal and mutated oligos : </li></ul>
  15. 16. Detection of Copy Number Changes <ul><li>Specific controls included in the chip and in each PCR group : </li></ul><ul><ul><li>Normalization : Chromosome 21 </li></ul></ul><ul><ul><li>Copy number change detection : Chromosome X </li></ul></ul><ul><ul><li>In each batch of hybridization, male and female controls are processed </li></ul></ul><ul><ul><li>Based on ratio of intensities of hybridization </li></ul></ul>
  16. 17. 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
  17. 18. ESSENTIAL EQUIPMENT REQUIRED FOR PROCESSING 1. Thermocycler: Applied Biosystem 9700 <ul><li>Patient DNA amplification (± 2h) </li></ul><ul><li>Fragmentation and Labelling (±1h45) </li></ul>
  18. 19. ESSENTIAL EQUIPMENT REQUIRED FOR PROCESSING 2.- Hybridization station: Tecan HS 4800™ Pro Hybridization Stations <ul><li>Hybridization of the amplified, fragmented and labelled DNAs </li></ul><ul><li>An “easy to use” software controlls all the process. </li></ul><ul><li>12 samples can be processed at the same time. </li></ul>
  19. 20. 3. Scanner: Innoscan 710A (Innopsys) <ul><li>Hybridized slides scanning (1 minute per slide) </li></ul><ul><li>Macros and software installed on the same computer for data analysis </li></ul>ESSENTIAL EQUIPMENT REQUIRED FOR PROCESSING
  20. 21. ESSENTIAL EQUIPMENT REQUIRED FOR PROCESSING 4. LIPOchip software: result analysis and report

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