LIPOChip

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 : 50-100μm

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

ATCGTAGC

AMPLIFICATION (PCR)(region of interest)

FRAGMENTATION

DNAse: random cutsAlkaline 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

N

N

N

M

M

M

N

N

N

Technical base

Experimental procedure

Oligonucleotide designMultiplex PCR design

DNA-chip design & printing

Hybridization optimization

SPECIFICATIONS

Mutations/SNPs to be analyzed

Image Capture and Software development

GENOTYPE

FUNCTION OF MUTANT PROBES VALUES

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1

1 28 55 82 109 136

Number of mutation

Fu

nc

tio

n o

f m

uta

nt

pro

be

s s

ign

al in

ten

sit

y

HOMOZYGOUS MUTANT PATIENTS HETEROZYGOUS MUTANT PATIENT HOMOZYGOUS NORMAL SUBJECTS

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:

TTTCTAGCAGGGGGAGGAGTTTGTTTCTAGCAGGCGGAGGAGTTTG

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

A

B

C

Multiplex amplification

Group 1 Group 2 Group 3 Group 4prom & ex 1 ex 3 ex 2 ex 5

ex 4 ex 6 ex 8 ex 7ex 9 & 10 ex 16 ex 11 ex 12

ex 13 & 14 APOB ex 26 ex 15 ex 17PCSK9 ex 2 PCSK9 ex 4 PCSK9 ex 7 & 10

Normal Sample

HTZ Mutated Sample

HMZ Mutated Sample

1000 500 ≈0

≈0 500 1000

In

In + Im

Inormal oligo (In)

Imutated oligo (Im)

Ratio 1 0.5 0

Genotypes computing

0

0,2

0,4

0,6

0,8

1

0 0,2 0,4 0,6 0,8 1

MNL005

• Based on intensity values of normal and mutated oligos:

Chr 21 Chr XNormal

exonDeleted

exon

Isample (male) 1000 500 1000 500

Icontrol (female) 1000 1000 1000 1000

Ratio 1 0.5 1 0.5

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

1 Amplification

7.5µl DNA (20ng/µl)

2Fragmentation

3Labelling

4Hybridization

5Results 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

DAY1 DAY 2

OVERLAPPING PROCESSES

56 ul

WORKFLOW

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