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Conformation Sensitive Capillary Electrophoresis Conformation Sensitive Capillary Electrophoresis
Daniel Ward Daniel Ward High Throughput Screening Facility (Wessex High Throughput Screening Facility (Wessex Salisbury) Salisbury)
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Confirmation Sensitive Capillary Confirmation Sensitive Capillary Electrophoresis Electrophoresis
wt
mut
wt
a b
Denature + Reanneal
Capillary Capillary electrophoresis electrophoresis
mut
heteroduplexes homoduplexes
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CSCE examples (BRCA1) CSCE examples (BRCA1)
wt
wt
1186 A>G
1048 del A
wt
wt
1445 T>A
1623 del TTAAA
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CSCE workflow CSCE workflow
n n Variable no of samples Variable no of samples n n Variable no of Variable no of PCRs PCRs (fragments) per sample (fragments) per sample n n 3 fluorescent labels (pooling x3) 3 fluorescent labels (pooling x3) n n 96 well plate format 96 well plate format
Sample PCR Pool products and dilute Electrophoresis
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Batch example Batch example
N
V
F
Frag 1 Frag 2
Frag 3 Frag 4
Frag 5 Frag 6
Pool 1
Frag 1 Frag 3 Frag 5
Pool 2
Frag 2 Frag 4 Frag 6
n n All All PCRs PCRs to work at one annealing temperature (61 to work at one annealing temperature (61º ºC) C) n n Any fragment can be labeled in any Any fragment can be labeled in any colour colour
Plate 1
Plate 2
Plate 3
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Universally tagged PCR Universally tagged PCR
Template DNA PCR
Product
GS1 GS2 M13F tag M13R tag
Sequencing reaction
M13F M13R
M13F = tgt aaa acg acg gcc agt M13R = cag gaa aca gct atg acc
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Template DNA PCR
Product
50bp buffer 50bp buffer
Assay Design: Standardised primer Assay Design: Standardised primer optimisation and design specification optimisation and design specification
F
F
GS1 GS2
M13FF
M13F = tgt aaa acg acg gcc agt M13R = cag gaa aca gct atg acc
M13F tag M13R tag M13R
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Assay Design: Standardised primer Assay Design: Standardised primer optimisation and design specification optimisation and design specification
Insert M13R
M13F
Cut site
M13F M13R
Approximate quantities in PCR
Plasmid 50,000 copies Primers 24,000 copies each
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Template DNA PCR
Product
50bp buffer 50bp buffer
Assay Design: Standardised primer Assay Design: Standardised primer optimisation and design specification optimisation and design specification
∞ 15 Hold
min 5 72 Final extension
sec 30 72 Primer extension x40 cycles
sec 30 61 GS annealing
sec 0 95 Denature
min 10 95 Taq activation
Cycle Time °C Step
F
F
GS1 GS2
US1F
US1 = gta gcg cga cgg cca gt US2 = cag ggc gca gcg atg ac
US1 tag US2 tag US2
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Assay Design: Standardised primer Assay Design: Standardised primer optimisation and design specification optimisation and design specification
C A G T A G C G A C G C G G G A C5’
5’G T A G C G C G A C G G C C A G T
US1F
US2
C A T C G C T G C G C C C T G
C A T C G C G C T G C C G G T
F
35bp 50bp 75bp 100bp
32bp
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Template DNA PCR
Product
50bp buffer 50bp buffer
Assay Design: Standardised primer Assay Design: Standardised primer optimisation and design specification optimisation and design specification
∞ 15 Hold
min 5 72 Final extension
sec 30 72 Primer extension x40 cycles
sec 30 61 GS annealing
sec 0 95 Denature
min 10 95 Taq activation
Cycle Time °C Step
F
F
GS1 GS2
US1F
US1 = gta gcg cga cgg cca gt US2 = cag ggc gca gcg atg ac
US1 tag US2 tag US2
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Primer optimisation Primer optimisation
Aim: Aim: clean trace with single peak within analysis window (1000 to clean trace with single peak within analysis window (1000 to 25000 RFU for 3730) 25000 RFU for 3730)
n n Primary optimisation [US1F]:[GS1]:[GS2] Primary optimisation [US1F]:[GS1]:[GS2] n n Determine fixed [US1F] (15 Determine fixed [US1F] (15 fmol fmol/ /μ μl reaction) l reaction) n n Determine titration ranges for individual optimisations Determine titration ranges for individual optimisations
[GS1] 3, 9, 27, 81 [GS1] 3, 9, 27, 81 fmol fmol/ /μ μl reaction l reaction [GS2] 40, 80, 160, 320 [GS2] 40, 80, 160, 320 fmol fmol/ /μ μl reaction l reaction
n n Individual fragment optimisation [ Individual fragment optimisation [GS1]:[GS2] GS1]:[GS2]
GS1 GS2
US1F
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BRCA2 Ex03
3
9
27
81
[GS1] fm
ol/µl rxn
[GS2] fmol/µl rxn 40 80 160 320
GS1 GS2
US1F
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BRCA2 Ex23
3
9
27
81
[GS1] fm
ol/µl rxn
[GS2] fmol/µl rxn 40 80 160 320
GS1 GS2
US1F
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BRCA2 Ex11E
3
9
27
81
[GS1] fm
ol/µl rxn
[GS2] fmol/µl rxn 40 80 160 320
GS1 GS2
US1F
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Optimisation Optimisation
92
100
100
Optimised (%)
15
29
2° (%)
92
84
64
1° (%)
1 46 46 BRCA2 Breast cancer
61 61 FBN1 Marfans
7 33 33 BRCA1 Breast cancer
3° (%)
Designed Fragments Gene Disease
n nFBN1 (61 fragments) FBN1 (61 fragments) n nDesign Design – – 1 week 1 week n nWait for primers! Wait for primers! n nOptimisation Optimisation 2 days 2 days n n1 1° ° Re Re design requirement design requirement – – 5 fragments (8%) 5 fragments (8%)
n nExperience gained over 2yrs used to refine process Experience gained over 2yrs used to refine process n nAutomated protocol Automated protocol
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Controls Controls n n Per fragment Per fragment
n n Mutation positive plasmid control Mutation positive plasmid control n n Mutation negative plasmid control Mutation negative plasmid control n n Polymorphism control Polymorphism control n n Water control Water control
n n Per run Per run n n 60.1 G>A reference control (x1) 60.1 G>A reference control (x1)
n n Per week Per week n n 60.1 G>A reference control (every capillary) 60.1 G>A reference control (every capillary)
60.1 G>A 60.1 G>A
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Post PCR processing Post PCR processing
n n Dilution ~1:50 in Dilution ~1:50 in 0.05mM EDTA 0.05mM EDTA
n n Mix products by colour (and/or size) Mix products by colour (and/or size) n n Currently 3 Currently 3 plex by colour (FAM, VIC, NED) plex by colour (FAM, VIC, NED) n n Potential up to 20 analyses per capillary (4x colour, 5x size) Potential up to 20 analyses per capillary (4x colour, 5x size)
n n Add size standard (0.02 Add size standard (0.02μ μl LIZ 500 per load) l LIZ 500 per load)
n n Loading volume 10 Loading volume 10 μ μl l n n Wax overlay Wax overlay
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BC103
BC108
BC118
BC111L
BC210B
BC112
Mixed products Mixed products
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wt
2196 G>A
2196 G>A + 2201 C>T + 2430 T>C
2201 C>T + 2430 T>C
Special cases Special cases Compound heterozygosity
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n n May give mobility shift (esp. May give mobility shift (esp. insdels insdels) )
Special cases Special cases Homozygotes Homozygotes
BRCA2 3396 A/A
BRCA2 3396 A/G
BRCA2 3396 G/G
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Sequential loading Sequential loading
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Dilution in water Dilution in 0.05 mM EDTA
Dilution of products Dilution of products
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Primary injection n th injection
Loss of resolution Loss of resolution
n nBreak down of the dynamic coating of the internal walls of Break down of the dynamic coating of the internal walls of the capillary the capillary n nResolution can be recovered by leaving the instrument in an Resolution can be recovered by leaving the instrument in an idle state (currently four hours) idle state (currently four hours)
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Validation Validation
n n CSCE setup using Sanger protocol (Davies CSCE setup using Sanger protocol (Davies et al et al 2006) 2006) which gives general conditions for CSCE which gives general conditions for CSCE
n n Generic Mutation Detection controls (GMD controls) Generic Mutation Detection controls (GMD controls) n n four different four different amplicons amplicons (20%, 40%, 60% and 80% GC rich) (20%, 40%, 60% and 80% GC rich) n n At 3 different positions in each At 3 different positions in each ampilcon ampilcon four different mutations four different mutations have been introduced have been introduced
n n 48 mutant controls and 4 wild type (WT) controls 48 mutant controls and 4 wild type (WT) controls
n n GMD set passed through the system and scored GMD set passed through the system and scored manually manually
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WT and 0 1 2
3 4 5
Validation Validation n nScoring system for CSCE traces Scoring system for CSCE traces
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Validation Validation n nTesting the Sanger protocol using Testing the Sanger protocol using the GMD controls gives a sensitivity of 98% (47/48); the only mutation not detected was 80.1G>A
nTwo main factors affecting detection capability are the GC content of the fragment and the position of the mutation in the fragment
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Conclusions Conclusions
n n We have developed an We have developed an ampilfication ampilfication system that allows: system that allows: n n Single PCR annealing temperature Single PCR annealing temperature n n Flexible fragment labelling Flexible fragment labelling n n Reduced primer cost Reduced primer cost n n Simple and informative optimisation Simple and informative optimisation
n n BRCA1 & 2 screen set up and operational (79 fragments) BRCA1 & 2 screen set up and operational (79 fragments) n n Marfans Marfans screen (FBN1) in optimisation (~60 fragments) screen (FBN1) in optimisation (~60 fragments) n n Next targets HNPCC and NOTCH1 Next targets HNPCC and NOTCH1
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Acknowledgements Acknowledgements CSCE CSCE n n Chris Mattocks Chris Mattocks NGRL (Wessex) NGRL (Wessex) n n Helen Davies Helen Davies Cancer Genome group, Sanger institute Cancer Genome group, Sanger institute n n Nick Owen Nick Owen NGRL (Wessex) NGRL (Wessex)
Generic and disease specific mutation controls Generic and disease specific mutation controls n n Helen White Helen White NGRL (Wessex) NGRL (Wessex) n n Vicky Hall Vicky Hall NGRL (Wessex) NGRL (Wessex) n n Gemma Potts Gemma Potts NGRL (Wessex) NGRL (Wessex)
HTSF HTSF n n Julie Sillibourne Julie Sillibourne n n Tracey Merrifield Tracey Merrifield n n Anne Anne Marie Coupe Marie Coupe n n Alison Skinner Alison Skinner n n Stacey Sandell Stacey Sandell