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Development of SYBR Green RT-qPCR to confirm small SNP array aberrations

Development of SYBR Green RT-qPCR to confirm small SNP array aberrations. Carolyn Dunn , Annabel Whibley, Lionel Willatt and Ingrid Simonic Cambridge. Overview of Array Results - 2007. 134 SNP Arrays - dev delay - congenital abnormalities.

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Development of SYBR Green RT-qPCR to confirm small SNP array aberrations

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  1. Development of SYBR Green RT-qPCR to confirm small SNP array aberrations Carolyn Dunn, Annabel Whibley, Lionel Willatt and Ingrid Simonic Cambridge

  2. Overview of Array Results - 2007 134 SNP Arrays - dev delay - congenital abnormalities 60% Normal Array Result 40% ? Del/dup Array Result Unsuitable for FISH: del <150kb or dup <1.5Mb (18%) FISH confirmatory studies (22%)

  3. Options for Confirmatory Studies • A second type of array • Re-analysis of whole genome • High set-up and running costs • MLPA • Able to multiplex • Cost of probe expensive for single family follow-up studies • RT-qPCR • Fluorescent Probes • SYBR Green • Low cost

  4. SYBR Green RT-qPCR Principles • Denaturation of DNA to produce ssDNA • Thermal Cycling: • Primers anneal to and extend target sequence • SYBR Green I binds to dsDNA and emits a fluorescent signal • As PCR amplification proceeds, (causing the amount of dsDNA to increase), the fluorescence signal increases proportionately ssDNA 3’ 5’ 3’ 5’ N.B. SYBR Green I binds all dsDNA (including primer-dimers and non-specific reaction products) – essential that primers are specific to target sequence

  5. SYBR Green RT-qPCR Strategy • Select target gene in UCSC/Ensembl • Export and repeat mask sequence • Primer design – Primer3 • SNP check (Manchester Diagnostic SNPCheck) and BLAST primer sequences • PCR reaction efficiency (90–110%) and precision (Rsq value >0.985)

  6. Overview of Primer Validations 24 sets of primers 2 taken from RTPrimerDB 22 designed using Primer3 2 Failed QC: reaction efficiency <90 or >110% or Rsq < 0.985 Passed QC 20 Passed QC Re-design Primers

  7. Proof-of-principle Study • Is this approach reliable and robust to use diagnostically? • Which real time PCR machine to use? ABI 7900 versus Rotor-Gene 65H0 • Ease of use, cost, consumables • Plates versus tubes on a rotor • 6 cases (5 duplications and 1 deletion) • Abnormal karyotype (4) or array result (2) • Confirmed by FISH

  8. Set-up and Analysis • 4 controls • GAPDH used as the reference gene • All reactions in triplicate - SD <0.18 • Each experiment replicated • Analysed using ∆∆Ct method and primer efficiency-corrected • Expected relative copy number • Normal: 1.0 (0.85-1.15) • Deletion: 0.5 (0.35-0.65) • Duplication: 1.5 (1.35-1.65) • Equivocal: 0.65-0.85 and 1.15-1.35

  9. Proof-of-principle Study Results

  10. SNP Array Follow-up Data (I) • 6 SNP array abnormalities followed-up by qPCR to date (5 patients) • 1 was not confirmed – within the ‘normal range’ • A high number of “calls” on the array analysis • One of the two array analysis packages highlighted this as an abnormality Summary of data from 2 qPCR experiments

  11. SNP Array Follow-up Data (II) • 6 SNP array abnormalities followed up to date (5 patients) • 1 was not confirmed - same CN as controls • 1 borderline equivocal/duplication result • primer pair failed QC • Re-design primers and repeat Summary of data from 2 qPCR experiments

  12. SNP Array Follow-up Data (III) • 6 SNP array abnormalities followed up to date (5 patients) • 1 was not confirmed - same CN as controls • 1 borderline equivocal/duplication result • primer pair failed QC • repeat with second set of primers • 4 confirmed (2 dels and 2 dups)

  13. SNP Array Follow-up Data (IV) • 300kb deletion (no suitable FISH clone) • 110kb deletion • 42kb duplication Summary of data from 2 qPCR experiments

  14. SNP Array Follow-up Data (VI) • 660kb ?dupXq27.1 (includes SOX3 gene) SOX3 X ?dupX

  15. Summary • Costs higher than first predicted as primer re-design required for some cases • Equivocal result • Primers that fail QC step • A copy number of 4 or greater may not be accurately detected • A promising option for verifying small array deletions or duplications

  16. Acknowledgments • Dr Lucy Raymond (Clinical Genetics, Addenbrooke’s Hospital) • Dr Martin Curran (Head of Molecular Diagnostic Microbiology Section, Addenbrooke’s Hospital)

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