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MICROARRAYS

MICROARRAYS. Eleanor Rattenberry Pre- reg Clinical Scientist Birmingham. Microarrays. Merge molecular diagnostics with traditional chromosome analysis Have the ability to detect any genomic imbalance including deletions, duplications, aneuploidies and amplifications . Clinical Service.

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MICROARRAYS

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  1. MICROARRAYS Eleanor Rattenberry Pre- reg Clinical Scientist Birmingham

  2. Microarrays • Merge molecular diagnostics with traditional chromosome analysis • Have the ability to detect any genomic imbalance including deletions, duplications, aneuploidies and amplifications

  3. Clinical Service Birmingham cytogenetic and molecular lab • ~6000 postnatal cytogenetic samples / year • ~3000 dev. delay / MR per year • Cytogenetics +/- FISH AND/OR • Molecular analysis (FRX – 0.4% pick-up rate) Microarray Service • Referral for microarray analysis by Clinical Geneticists • ~300 retrospective cases in backlog • ~250 new cases per year Candidates for array analysis • Previously normal cytogenetics / FISH / molecular testing • Apparently balanced rearrangement with phenotype

  4. Array platforms used currently • BlueGnome CytoChip 1.1 1Mb BAC array • Frontline clinical service array • Affymetrix 500K SNP array • European Cytogenetic Initiative (ECI) project

  5. 1Mb BAC array results *According to Redon / DGV

  6. Classification of non-CNV copy number changes 1Mb BAC array results Total running abnormality rate >24%

  7. Affymetrix 500K SNP arrays • Affymetrix funded European Cytogenetic Initiative (ECI) • Birmingham, UK • Nijmegen, Netherlands • Tuebingen, Germany • Clinical suitability: specificity and sensitivity • 120 patient / 240 parent trio assembly • 40 “known” abnormals • Project-specific reference set • Analysis: CNAT vs CNAG (ongoing) • 8/40 (20%) de novo imbalance (>500Kb)

  8. Overview • DNA quality • Affymetrix 500K SNP array • Background • Technique • Case study • BlueGnome 1Mb array • Background • Technique • Case study • Validation of array findings

  9. DNA Quality

  10. DNA Sample Quality • High molecular weight • As low level of contaminants as possible • Proteins • RNA • Phenols • Alcohols • Salts

  11. DNA Quality Control 1 2 3 4 5 1kb ladder 260/280 - 1.8-1.9 260/230 - 1.8-2.0 √ √ × ? ?

  12. Affymetrix SNP Arrays

  13. Single Nucleotide Polymorphisms (SNPs) • The most abundant form of genetic variation in the human genome • Estimated to be ~11 million SNPs spread throughout the genome

  14. Arrays Available 5 micron = 1/200th of a millimeter

  15. Applications of Technology • In house • Copy number variation (CNV) • Linkage (autozygosity mapping) • Other applications • Cancer studies • Expression

  16. SNP C / A PM Allele A MM Allele A PM Allele B MM Allele B Probe Quartets Genomic Sequence GTACTCAATGATCAGCT TAGCCATCGGTAN ATCGGTAGCCAT G CATGAGTTACTA ATCGGTAGCCAT C CATGAGTTACTA Probe Quartet ATCGGTAGCCAT T CATGAGTTACTA ATCGGTAGCCAT A CATGAGTTACTA

  17. Allele ‘A’ Allele ‘B’ ATCGGTAGCCAT G CATGAGTTACTA PM Allele ATCGGTAGCCAT C CATGAGTTACTA MM Allele ATCGGTAGCCAT T CATGAGTTACTA PM Allele MM Allele ATCGGTAGCCAT A CATGAGTTACTA Patient: homozygous CC TAGCCATCGGTA C /C GTACTCAATGATCAGCT SNP

  18. Allele ‘A’ Allele ‘B’ ATCGGTAGCCAT G CATGAGTTACTA PM Allele ATCGGTAGCCAT C CATGAGTTACTA MM Allele ATCGGTAGCCAT T CATGAGTTACTA PM Allele MM Allele ATCGGTAGCCAT A CATGAGTTACTA Patient: heterozygous CA TAGCCATCGGTA C / A GTACTCAATGATCAGCT SNP

  19. Allele ‘A’ Allele ‘B’ ATCGGTAGCCAT G CATGAGTTACTA PM Allele ATCGGTAGCCAT C CATGAGTTACTA MM Allele ATCGGTAGCCAT T CATGAGTTACTA PM Allele MM Allele ATCGGTAGCCAT A CATGAGTTACTA Patient: homozygous AA TAGCCATCGGTA A/ A GTACTCAATGATCAGCT SNP

  20. 14 Quartets were evaluated and the 6 best performing were chosen to represent each SNP These 6 quartets interrogate each SNP either the Forward and/or on the Reverse strand. Some probe sets have all probes on one strand. 24 probes total are used to interrogate a single SNP Quartet 500K Probe Array Tiling

  21. Distributed Tiling Strategy The PM-MM probe pairs are distributed across the array

  22. Introduction to Affymetrix 500K Assay

  23. 500K Assay • 2 arrays • Made up of different SNPs, process is the same apart from use of different enzymes: • NspI (262K SNPs) • StyI (238K SNPs)

  24. Assay Overview DAY ONE Restriction Digestion High Quality Genomic DNA (250ng) Cut Cut Cut Cut 200 – 1100 bases Adapter Ligation SinglePrimerAmplification DAY FOUR DAY TWO DAY THREE Hyb, Wash, and Stain. Scan and Data Analysis Purification of PCR Products Fragmentation and Labeling

  25. Pre-PCR Clean Area Template-free hood Reaction setup (master mixes) PCR Staging Area Low copy template lab Non -amplified template added to reactions Main Lab Contaminated with PCR product No PCR staging room re-entry after working here Laboratory Setup

  26. Genomic DNA Preparation • Starting Material: 500ng high quality genomic DNA (QC as prev explained) • 250 ng for the NspI digest array • 250 ng for the StyI digest array • DNA is diluted to 50ng/ul in reduced EDTA TE buffer - Elevated EDTA interferes with downstream reactions

  27. Genomic DNA NspI NspI RE digestion SNP Restriction Enzyme Digests • NspI and StyI are six base cutters with wobble: NspI StyI • 2 hour digest at 37oC, inactivate enzyme at 65oC Recommendation: Run a positive and negative control R = purines C/T Y = pyrimidines A/G W = A/T

  28. Adaptor Ligation Adaptor Ligation • NspI adapter has a standard sequence • Sty I adapter is made up of a pool to account for restriction site base non-specificity • 3 hour reaction at 16oC (DNA ligase), 20 minute enzyme inactivation at 70oC

  29. PCR Complexity Reduction PCR Amplify 200 – 1100 bp fragments • 3 reactions per sample • Yield necessary for fragmentation is 90ug • Enzyme is Titanium Taq (hot start taq, uses Ab) • GC Melt is used as a PCR enhancer • Thermal cycler program: • Complexity reduction PCR

  30. PCR Products on a 2% TBE Gel PCR products of 200-1100 bp on 2% TBE Gel

  31. PCR Clean Up • Clonetech DNA Amplification Clean Up Kit • Requires a ~600 mbar vacuum

  32. PCR Quantitation • Very important to be accurate for the next step (Fragmentation) • Measure OD within linear range of your instrument • Run 3 independent dilutions to assure accuracy • Normalize PCR product to 2ug/uL in EB buffer • Goal: After clean up the yield for each PCR product > 90 µg

  33. Fragmentation Fragmentation • 90ug of purified PCR product is fragmented (DNase I) • Expected size for fragmentation is <180 bp • 35 minute reaction at 37oC followed by a 15 minute heat inactivation at 95oC • Check for proper fragmentation using a 4% TBE gel

  34. DNA Target Post Fragmentation 4% TBE Agarose Gel

  35. Labelling Labelling • Prepare the Labelling master mix on ice • Biotin-labelled deoxynucleotide • Terminal Deoxynucleotidyl Transferase • 5x Terminal Deoxynucleotidyl Transferase Buffer • Incubate at 37oC for 4 hours followed by a 15 minute heat inactivation at 95oC

  36. Hybridisation • 190 µl hybridization cocktail master mix added to 70 µl end-labeled DNA Target • Hybridisation cocktail: • Oligonucleotide Control Reagent • B2 oligo • 4 spike controls • Blocking agents – Human Cot-1 & HSDNA • TMACL– increases Tm of A-T bonds • DMSO – decreases Tm of G-C bonds

  37. Denaturation • Disassociate the DNA strands • 95-99oC for 10 minutes • Ice for 10 seconds*(longer or shorter times on ice decrease the call rate) • 49oC for 1 minute • Keep samples at 49oC until ready to add to array • Hybridize for 16-18 hours @ 49oC and 60 rpm

  38. Staining, Washing and Scanning • Three stain buffers: • Stain 1 – Streptavidin R-Phycoerythrin • Stain 2 - Anti-streptavidin monoclonal antibody • Stain 3 - Array Holding Buffer • Two wash buffers: • Wash A – low stringency • Wash B – high stringency

  39. SAPE SAPE SAPE SAPE SAPE SAPE SAPE SAPE SAPE SAPE SAPE SAPE SAPE SAPE SNP probe = 25 bases GLASS WAFER

  40. Data Analysis

  41. Data Analysis • Genechip Data Analysis Software (GDAS) • Genotypes each SNP • Provides a percentage call rate • Sample mismatch (50 SNPs common on each 250K array) • Sample identity – confirms parental samples • Copy Number Analysis Tool • Determines copy number of each SNP against a control population • Determines LOH against a control population

  42. Case Study

  43. Case 1 • 2yrs old • Abnormal triple test antenatally • ? Trisomy 18 (no invasive test) • Seizures by 6/52 – complex, partial • Atrial Septal Defect • Developmental delay: not sitting at 8/12 • Dysmorphic • Metopic ridge and trigonocephaly • microcephaly • Broad depressed nasal bridge / broad tip • Mildly upslanting palpebral fissures • Carp-shaped mouth with bowed lower lip

  44. Case 1: del(1)(q43q44) Parents normal

  45. Case 1: del(1)(q43q44) • del(1)(q42qter) • Merritt (Am J Med Genet, 2007) • 7 cases reviewed • Phenotypic / physical overlap

  46. Array-CGH Judith Walker Pre-reg clinical scientist Birmingham

  47. BlueGnome 1Mb enhanced Specifically designed to meet the requirements of diagnostic clinical aCGH Assay: Easy to implement Highly automatable Results: Reliable Reproducible Easy to interpret

  48. BlueGnome 1Mb enhanced Pt Ref Pt Ref • Duplicate sub arrays • Dye swap • Extensive replication: • Backbone clones x2 (ds x4) • Disease clones x3 (ds x6)

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