Transcript Wadsworth Center

Invader/Luminex/Microarray/MLPA
CFTR
Carlos A. Saavedra-Matiz, MD
Newborn Screening Program
Wadsworth Center
New York State Department of Health
June 30, 2011
APHL-CDC
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Dried Blood Spot (DBS)
“Guthrie” Card
S&S® 903™ Cotton Paper
- 3.1 mm DBS ~ 3.1 uL whole blood.
- Benefits:
- Rapid absorption (~ 10 seconds)
- Easy transportation
- Blood constituents “easily” eluted
- Typeable DNA from 3-15 years.
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1043 articles in PubMed 1971-2011
- 369,000 Google “hits”
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SAMPLES ARE PUNCHED INTO 96-WELL PLATES
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Enzyme-Linked Immunosorbent Assay (ELISA)
•Heat denaturation typically 95 degrees C.
•Provides sufficient energy to break hydrogen bonds.
POLYMERASE CHAIN REACTION
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•Primer annealing typically 55-65 degrees C.
•Takes advantage of base complementarity.
POLYMERASE CHAIN REACTION
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•Polymerization at 72 degrees C.
•Requires availability of Thermus aquaticus
DNA polymerase (heat stable).
POLYMERASE CHAIN REACTION
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•After the first cycle, there are 2 copies of the
original double helix.
•Continue cycling….
POLYMERASE CHAIN REACTION
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•And so on…for 30-35 cycles
•Result is billion-fold amplification of target.
POLYMERASE CHAIN REACTION
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- The Cystic Fibrosis Transmembrane conduction Regulator gene (CFTR) has
27 exons. Its locus spans 250 kb at 7q31.3
- CFTR encodes a membrane-bound chloride ion transport protein of 1,480
amino acids and multiple alleles.
- ΔF508, a deletion of a phenylalanine codon in position 508, is the most
common mutation (~70%)
Cystic Fibrosis Algorithm
Elevated IRT (80% false positive rate)
(top 5%)
Second test to confirm in-house
Additional predictors in development
~ 13,000 per year
Run 39 mutation + Poly + 5T + Reflex assay
1 or 2 mutations
IRT top 0.1%
Refer to CF Center for follow-up
(Evaluation, Sweat Chloride test & Genetic
Counseling)
0 mutations
Screen
Negative
 ‘Homebrew’ Manual/Automated Extraction
 3mm dried blood spot

100ul Total Volume

7 Tips/ Sample

Standard 96 Well Plate

Average Yield: 4-5ng/ul
Invader assay
Hsu, T. M. et al. Clin Chem 2001;47:1373-1377
Copyright ©2001 American Association for Clinical Chemistry
Site of
Cleavage
Site of
Cleavage
Primary
Reaction
Secondary
Reaction
Total: 42 ACMG/ACOG 23
and V520F, 3876delA,
394delTT, R347H, I148T,
1078delT, 3905insT, S549N,
Y122X, Y1092X
S549R(T>G), 2183AA>G,
S549R(A>C), D1152H, 3849 +
4A>G,E60X, Q493X, D1270N,
Y1092X(C>G)
Nucleic Acid
Extraction and
Purification
A optimal input
quantity of 50ng (range
of 10 ng to 1.5 ug) per
sample is required to
perform the assay.
Step 1 - Multiplex
PCR Reaction will
make multiple copies of
multiple DNA targets
within the CFTR gene.
Step 2 - Amplicon
Treatment
Enzymatic treatment
of amplified PCR
products cleaves
unused reagents
(primers and dNTPs)
left over after PCR.
Step 3 - Allele-specific primer
extension (for CF)
The amplified DNA is mixed
with short sequences (TAG
primers) of DNA specific to
each target. If the target is
present, the primer will bind
and will be lengthened through
a process called Allele specific
extension. During this
extension, a reporter label is
incorporated.
Step 4 - Bead Hybridization
Color-coded beads are added to
identify the tagged primers.
Attached to each differently colored
bead is an anti-TAG sequence
specific to one of the extended TAG
primers. Each anti-TAG only binds
to the complementary TAG
sequence on the primer.
Step 5 - Addition of
Reporter Molecule
The reporter solution is the
Streptavidin, R-Phycoerythrin
conjugate and will be used to
detect the target.
Step 6 - Data Acquisition on
Luminex Analyser
Samples are then placed in a
Luminex xMAP® instrument where
beads are read and analyzed by lasers.
The lasers identify the color of the
bead and the presence or absence of
the labeled target. For each sample,
these signals are interpreted by the
xTAG Data Analysis Software to
determine whether the wild-type
and/or mutant alleles for each of the
variations have been detected
Each complete Liquid Handling System is capable of 1200 DNA extractions per 8 hour day.
Real Time PCR
Taqman
•
Reporter/Quencher
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5’ Nuclease Activity
•
Probe Cleavage
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Sequence Specific
•
Multiplex Capability
TREC AMPLIFICATION PLOT
RNASE P AMPLIFICATION PLOT
ADA
ADA
ADA
RNaseP
TREC
Normal
Mutation
Present
FRET fluorescent resonance
energy transfer theory
Roche
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Microarray
MLPA
(Multiplex Ligation-dependent Probe Amplification)
MLPA® is a rapid, high-throughput
technique for copy number quantification.
Applications include:
•Congenital & Hereditary Disorders
•Tumours Profiling
•Methylation profiling
•Quantification of mRNA
•Angelman
•Beckwith-Wiedemann Syndrome (BWS),
Russell-Silver Syndrome (RSS)
•DiGeorge syndrome, Velocardiofacial
syndrome (VCFS, Cat eye syndrome (CES)
•
•Down syndrome, Edwards syndrome, Patau
syndrome
•Fragile X
Achondroplasia:
- Prominent forehead
- Macrocephaly
- Jaw prognatism
- Short limbs/neck
- Midfacial hypoplasia
- Most common form of short-limb dwarfism
- Mutations in the FGFR3 (fibroblast growth factor receptor-3) gene
- ~ 90% de novo
- Autosomal dominant
- FGFR3 is a negative regulator of chondrocyte
proliferation and differentiation in the growth plate
Muchas Gracias!
Soledad Matiz de Saavedra
1924-2010