1. Carolyn Dunn, Annabel Whibley, Lionel Willatt and Ingrid Simonic
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 3’
ssDNA 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)
Each primer 15-30bp
Tm of two primers within 2ºC of each other
GC content 45-55%
Try to avoid 3’ terminal T
Product size

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 ( )
Deletion: 0.5 ( )
Duplication: 1.5 ( )
Equivocal: and

9. Proof-of-principle Study Results
Abnormality
qPCR confirmation
dup(2)(q14.2q14.2)
Yes
dup(7)(q11.23q11.23)
dup(5)(p15.3p15.3)
dup(12)(q24.32q24.32)
dup(5)(p14.3p14.3)
Deletion
del(5)(p14.3p14.3)
Initially some results were not reliable – duplicate reps were replaced with triplicate
Reaction volume was reduced to 10ul but some SDs were high so back to 20ul
Conflicting result:
Duplication by karyotype and FISH but deletion by qPCR!
Patient with a deletion of same gene showing deletion by qPCR

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 A copy number of 4 or greater may not be accurately detected
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)