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
60% Normal
Array Result
40% ? Del/dup
Array Result
FISH
confirmatory
studies (22%)
Unsuitable for
FISH: del <150kb
or dup <1.5Mb
(18%)
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
I. Fluorescent Probes
II. SYBR Green
• Low cost
SYBR Green RT-qPCR
Principles
1. Denaturation of DNA to produce
ssDNA
2. Thermal Cycling:
• Primers anneal to and extend
target sequence
3. SYBR Green I binds to dsDNA
and emits a fluorescent signal
4. As PCR amplification proceeds,
(causing the amount of dsDNA to
increase), the fluorescence signal
increases proportionately
3’
3’
N.B. SYBR Green I binds all dsDNA (including primer-dimers and
non-specific reaction products) – essential that primers are
specific to target sequence
ssDNA
5’
5’
SYBR Green RT-qPCR
Strategy
1. Select target gene in UCSC/Ensembl
2. Export and repeat mask sequence
3. Primer design – Primer3
4. SNP check (Manchester Diagnostic
SNPCheck) and BLAST primer sequences
5. PCR reaction efficiency (90–110%) and
precision (Rsq value >0.985)
Overview of Primer Validations
24 sets of primers
2 taken from
RTPrimerDB
Passed QC
22 designed using
Primer3
2 Failed QC:
reaction efficiency
<90 or >110% or
Rsq < 0.985
Re-design Primers
20 Passed QC
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
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 efficiencycorrected
• 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
Proof-of-principle Study Results
Abnormality
qPCR confirmation
dup(2)(q14.2q14.2)
Yes
dup(7)(q11.23q11.23)
Yes
dup(5)(p15.3p15.3)
Yes
dup(12)(q24.32q24.32)
Yes
dup(5)(p14.3p14.3)
Deletion
del(5)(p14.3p14.3)
Yes
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
?del 14q21.3
1.50
Relative CN
1.09
1.00
0.98
0.50
0.00
Control average
Patient average
Sample
Summary of data from 2 qPCR experiments
• One of the two array analysis
packages highlighted this as an
abnormality
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
?dup12q21.1-q21.2
1.28
Relative CN
1.50
1.02
1.00
0.50
0.00
Control average
Patient average
Sample
Summary of data from 2 qPCR experiments
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)
SNP Array Follow-up Data (IV)
1.50
Relative CN
• 300kb deletion (no
suitable FISH clone)
?del7q11.21
0.99
1.00
0.39
0.50
0.00
Control average
Patient average
Sample
?del9q33.1
1.50
Relative CN
• 110kb deletion
1.07
1.00
0.37
0.50
0.00
Control average
Patient average
Sample
• 42kb duplication
?dup2q37.3
Relative CN
2.00
1.50
1.58
1.07
1.00
0.50
0.00
Control average
Summary of data from 2 qPCR experiments
Patient average
Sample
SNP Array Follow-up Data (VI)
• 660kb ?dupXq27.1 (includes SOX3 gene)
SOX3
Relative
RelativeCopy
CopyNumber
Number
8.0
8
7.0
6.0
7
6
5.0
4.0
4
5
33.0
22.0
11.0
00.0
X
?dupX
Female
C (n=5)
Female
C
(n=9)
Male
Male
C C (n=5)XXXXY
(n=8)
Sample
Sample
Patient
X X
Patient
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
Acknowledgments
• Dr Lucy Raymond (Clinical Genetics,
Addenbrooke’s Hospital)
• Dr Martin Curran (Head of Molecular Diagnostic
Microbiology Section, Addenbrooke’s Hospital)