Development of SYBR Green RT-qPCR to confirm small SNP array aberrations
Carolyn DunnCarolyn Dunn, Annabel Whibley, Lionel , Annabel Whibley, Lionel Willatt and Ingrid SimonicWillatt and Ingrid Simonic
Cambridge
Overview of Array Results - 2007
134 SNP Arrays - dev delay
- congenital abnormalities
Unsuitable for FISH: del <150kb
or dup <1.5Mb (18%)
60% Normal Array Result
FISH confirmatory
studies (22%)
40% ? Del/dup Array Result
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 ProbesII. SYBR Green
• Low cost
SYBR Green RT-qPCR
Principles1. Denaturation of DNA to produce
ssDNA2. 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’ 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
ssDNA
SYBR Green RT-qPCR
Strategy1. 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 sequences5. PCR reaction efficiency (90–110%) and
precision (Rsq value >0.985)
Overview of Primer Validations
24 sets of primers
2 taken from RTPrimerDB
22 designed using Primer3
Re-design Primers
Passed QC 20 Passed QC2 Failed QC: reaction efficiency <90 or >110% or Rsq < 0.985
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 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
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’
?del 14q21.3
0.981.09
0.00
0.50
1.00
1.50
Control average Patient average
Sample
Re
lative
CN
Summary of data from 2 qPCR experiments
• A high number of “calls” on the array analysis
• 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
1.02
1.28
0.00
0.50
1.00
1.50
Control average Patient average
Sample
Re
lati
ve
CN
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)
• 300kb deletion (no suitable FISH clone)
• 110kb deletion
• 42kb duplication
?del7q11.21
0.99
0.39
0.00
0.50
1.00
1.50
Control average Patient average
Sample
Re
lativ
e C
N
?dup2q37.3
1.07
1.58
0.00
0.50
1.00
1.50
2.00
Control average Patient average
Sample
Re
lativ
e C
N
?del9q33.1
1.07
0.37
0.00
0.50
1.00
1.50
Control average Patient average
Sample
Re
lativ
e C
N
Summary of data from 2 qPCR experiments
SNP Array Follow-up Data (VI)• 660kb ?dupXq27.1 (includes SOX3 gene)
0
1
2
3
4
5
6
7
8
Female C (n=5) Male C (n=5) Patient X
Sample
Re
lativ
e C
op
y N
um
be
r
X ?dupX0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
Female C(n=9)
Male C(n=8)
XXXXY Patient X
Sample
Rel
ativ
e C
opy
Num
ber
SOX3
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)
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