Presentation is loading. Please wait.

Presentation is loading. Please wait.

Invader/Luminex/Microarray/MLPACFTR June 30, 2011 APHL-CDC Carlos A. Saavedra-Matiz, MD Newborn Screening Program Wadsworth Center New York State Department.

Similar presentations


Presentation on theme: "Invader/Luminex/Microarray/MLPACFTR June 30, 2011 APHL-CDC Carlos A. Saavedra-Matiz, MD Newborn Screening Program Wadsworth Center New York State Department."— Presentation transcript:

1 Invader/Luminex/Microarray/MLPACFTR June 30, 2011 APHL-CDC Carlos A. Saavedra-Matiz, MD Newborn Screening Program Wadsworth Center New York State Department of Health

2 Wadsworth Center

3 Dried Blood Spot (DBS) “Guthrie” Card - S&S ® 903 ™ Cotton Paper mm DBS ~ 3.1 uL whole blood. - Benefits: - Rapid absorption (~ 10 seconds) - Easy transportation - Blood constituents “easily” eluted - Typeable DNA from 3-15 years articles in PubMed ,000 Google “hits”

4 SAMPLES ARE PUNCHED INTO 96-WELL PLATES Wadsworth Center

5

6 1234

7

8 Enzyme-Linked Immunosorbent Assay (ELISA)

9 Heat denaturation typically 95 degrees C. Provides sufficient energy to break hydrogen bonds. POLYMERASE CHAIN REACTION Wadsworth Center

10 Primer annealing typically degrees C. Takes advantage of base complementarity. POLYMERASE CHAIN REACTION Wadsworth Center

11 Polymerization at 72 degrees C. Requires availability of Thermus aquaticus DNA polymerase (heat stable). POLYMERASE CHAIN REACTION

12 Wadsworth Center After the first cycle, there are 2 copies of the original double helix. Continue cycling…. POLYMERASE CHAIN REACTION

13 Wadsworth Center And so on…for cycles Result is billion-fold amplification of target. POLYMERASE CHAIN REACTION

14 - The Cystic Fibrosis Transmembrane conduction Regulator gene (CFTR) has 27 exons. Its locus spans 250 kb at 7q 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%)

15 Cystic Fibrosis Algorithm Elevated IRT (80% false positive rate) (top 5%) Second test to confirm in-house Run 39 mutation + Poly + 5T + Reflex assay Refer to CF Center for follow-up (Evaluation, Sweat Chloride test & Genetic Counseling) 1 or 2 mutations0 mutations Additional predictors in development ~ 13,000 per year Screen Negative IRT top 0.1%

16  ‘ Homebrew’ Manual/Automated Extraction  3mm dried blood spot  100ul Total Volume  7 Tips/ Sample  Standard 96 Well Plate  Average Yield: 4-5ng/ul

17 Copyright ©2001 American Association for Clinical Chemistry Hsu, T. M. et al. Clin Chem 2001;47: Invader assay

18 Site of Cleavage

19 Primary Reaction Secondary Reaction

20 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, A>G,E60X, Q493X, D1270N, Y1092X(C>G)

21

22 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

23

24 Each complete Liquid Handling System is capable of 1200 DNA extractions per 8 hour day.

25 Real Time PCR Reporter/Quencher 5’ Nuclease Activity Probe Cleavage Sequence Specific Multiplex Capability Taqman

26 TREC AMPLIFICATION PLOTRNASE P AMPLIFICATION PLOT ADA

27 RNaseP TREC

28 Normal Mutation Present FRET fluorescent resonance energy transfer theory Wadsworth Center Roche

29

30 Microarray

31

32 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

33 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

34 Muchas Gracias ! Soledad Matiz de Saavedra


Download ppt "Invader/Luminex/Microarray/MLPACFTR June 30, 2011 APHL-CDC Carlos A. Saavedra-Matiz, MD Newborn Screening Program Wadsworth Center New York State Department."

Similar presentations


Ads by Google