Conformation Sensitive Capillary Electrophoresis Conformation Sensitive Capillary Electrophoresis

Daniel Ward Daniel Ward High Throughput Screening Facility (Wessex High Throughput Screening Facility (Wessex­ ­Salisbury) Salisbury)

Confirmation Sensitive Capillary Confirmation Sensitive Capillary Electrophoresis Electrophoresis

wt

mut

wt

a b

Denature + Re­anneal

Capillary Capillary electrophoresis electrophoresis

mut

heteroduplexes homoduplexes

CSCE examples (BRCA1) CSCE examples (BRCA1)

wt

wt

1186 A>G

1048 del A

wt

wt

1445 T>A

1623 del TTAAA

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

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

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

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

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

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

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

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

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

BRCA2 Ex03

3

9

27

81

[GS1] fm

ol/µl rxn

[GS2] fmol/µl rxn 40 80 160 320

GS1 GS2

US1F

BRCA2 Ex23

3

9

27

81

[GS1] fm

ol/µl rxn

[GS2] fmol/µl rxn 40 80 160 320

GS1 GS2

US1F

BRCA2 Ex11E

3

9

27

81

[GS1] fm

ol/µl rxn

[GS2] fmol/µl rxn 40 80 160 320

GS1 GS2

US1F

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

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

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

BC1­03

BC1­08

BC1­18

BC1­11L

BC2­10B

BC1­12

Mixed products Mixed products

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

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

Sequential loading Sequential loading

Dilution in water Dilution in 0.05 mM EDTA

Dilution of products Dilution of products

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)

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

WT and 0 1 2

3 4 5

Validation Validation n nScoring system for CSCE traces Scoring system for CSCE traces

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

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

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