Temperature Gradient Capillary Electrophoresis Using the Reveal

Temperature Gradient Capillary Electrophoresis Using the Reveal

Temperature Gradient Capillary Electrophoresis Using the Reveal Genetic Mutation Discovery System (( Spectrumedix
Spectrumedix ) JK Campbell
JK Campbell Ø Ø Overview of Spectrumedix System Ø Ø Summary of our experience using it for mutation scanning at Guy’s
mutation scanning at Guy’s Reveal Genetic Mutation Discovery System (( Spectrumedix
Spectrumedix ) Ø Ø
Temperature gradient capillary electrophoresis (TGCE) l l
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Fragments with different melting properties can be analysed together Multiple DNA fragments can be analysed together Uses ethidium bromide to detect DNA l l
No need for fluorescent primers 96 Capillaries Ø Ø Specific software to compare peak traces and identify mutations Ø Ø Can also be used for genotyping and sequencing
sequencing Ø Ø
Reveal Genetic Mutation Discovery System Final Temp Tm amplicon B and C p
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Tm amplicon A T
Initial Temp em
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Time A Time B mutation start normal mutation normal mutation Time A normal mutation normal Time B Revelation Mutation Detection Software Raw data Normalised data Mutation Report
Revelation Mutation Detection Software
Revelation Mutation Detection Software Revelation Mutation Detection Software
Revelation Mutation Detection Software Revelation Mutation Detection Software
Revelation Mutation Detection Software Revelation Mutation Detection Software
Revelation Mutation Detection Software Sensitivity and Specificity TGCE CSCE
CSCE True positive 84 89 True negative 54 73 False positive 26 6 False negative 4 7 Fails 8 1 Sensitivity 95% 93% Specificity 68% 92% Some mutations give very subtle peak traces Dystrophin ex44 c.6438+2T>A
BRCA1 ex13 4327C>T Revelation software doesn’t detect all peak changes
all peak changes Some mutations are only detected at one temperature range BRCA1 ex11I 3119G>A 50­60 o C 35­45 o C
BRCA2 ex11PQ 4656T>C Some fragments are not suitable for TGCE Dystrophin ex 26 Poly A tract COL4A5 ex 26 Poly T tract c.2041+1G>T
Using Tagged primers can alter peak traces NO TAG Dystrophin Ex59 Dystrophin Ex75 TAG
Other problems Ø Ø Peak strengths Ø Ø Reproducibility Ø Ø General Maintenance Ø Ø Technical support
Technical support Summary of mutation screening using the Spectrumedix at Guy’s Ø Ø Congenital Muscular Dystrophy l l
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POMT1, POMT2, POMGnT1, LARGE, Fukutin LAMA2 Ø Ø Hereditary Spastic Paraplegia l l
Spastin (SPG4) Ø Ø Hereditary Breast and Ovarian Cancer l l
BRCA1 and BRCA2 Ø Ø Alports l l
COL4A5 Ø Ø DMD/BMD l l
Dystrophin
Dystrophin Summary of mutation screening using the Spectrumedix at Guy’s Ø Ø Congenital Muscular Dystrophy l l
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POMT1, POMT2, POMGnT1, LARGE, Fukutin LAMA2 Ø Ø Hereditary Spastic Paraplegia l l
Spastin (SPG4) Ø Ø Hereditary Breast and Ovarian Cancer l l
BRCA1 and BRCA2 Ø Ø Alports l l
COL4A5 Ø Ø DMD/BMD l l
Dystrophin
Dystrophin Summary Ø Ø Sensitivity is comparable to other techniques Ø Ø Specificity is low Ø Ø Software cannot be relied on for mutation calling Ø Ø Best for diseases with l l
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Low level of polymorphism A high mutation detection rate Small numbers of samples
Small numbers of samples Acknowledgements Ø Ø Helen White Ø Ø Annabel Whibley Ø Ø Judith Pagan Ø Ø Rachael Mein Ø Ø Caroline Godfrey Ø Ø David Moore
David Moore