Post on 11-May-2015
description
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 : 50-100μm
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