LIPOChip

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Progenika

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

Santé & Médecine

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? ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

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 ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

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 ,[object Object],[object Object],[object Object],Allele-Specific Oligonucleotides: TTTCTAGCAGG G GGAGGAGTTTG TTTCTAGCAGG C GGAGGAGTTTG 11nt wt mut 11nt

Oligonucleotide design ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

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

Multiplex PCR Design ,[object Object],A B C

Detection of Copy Number Changes ,[object Object],[object Object],[object Object],[object Object],[object Object]

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 ,[object Object],[object Object]

ESSENTIAL EQUIPMENT REQUIRED FOR PROCESSING 2.- Hybridization station: Tecan HS 4800™ Pro Hybridization Stations ,[object Object],[object Object],[object Object]

3. Scanner: Innoscan 710A (Innopsys) ,[object Object],[object Object],ESSENTIAL EQUIPMENT REQUIRED FOR PROCESSING

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

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