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

2. The first DNA chip with CE MARK DETECTION OF THE MOST FREQUENT FH MUTATIONS AND COPY NUMBER CHANGES OF THE LDLR GENE

4. Biochip Chip/Array : DNA fragments (15-60bp) printed (spotted) on a solid support (glass) in an ordered way

5. Size of one spot : 50-100 μm 60-75mm Biochip

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

8. N M Homozygous donor Heterozygous donor Technical base N N N M M M N N N

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

12. Multiplex PCR Design M1 M2 M3 Exon 1 M4 M5 Exon 6

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

21. ESSENTIAL EQUIPMENT REQUIRED FOR PROCESSING 4. LIPOchip software: result analysis and report