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DON LOVE DIAGNOSTIC GENETICS
FISHKARYOTYPING
SEQUENCING
PROTEIN MODELLING
QUANTITATION
CNV
GROWTH DEMANDS REDESIGN:A CASE STUDY OF MOLECULAR GENETICS
GENES IMPLICATED IN LONG QT SYNDROMEGENES IMPLICATED IN LONG QT SYNDROMEClassification Gene Chromosome Ion Channel Type Frequency Types of Mutation
LQT1 KCNQ1
KCNH2
SCN5A
ANK2
KCNE1
KCNE2
KCNJ2
CACNA1c
Cav3
SCN4B
LQT2
Mainly missense, insertion, deletions
30-50%IKs α-subunit11
7
3
4
21
21
LQT3
2
Mainly missense and insertions, deletions
12
25-45%
3
IKr α-subunit
Na+ α-subunit
Adaptor protein
IKs β-subunit
LQT4
IKr β-subunit
K+ channel
Mainly missense and deletions
Ca2+ channel
Membrane domain
5-10%
<1
<1
<1
LQT5
<1
<1
11
Missense
Missense and deletions
Missense
Missense and deletions
Na+ β4-subunit
<1
LQT6
LQT7
<1
Missense
Missense
Missense
LQT8
LQT9
LQT10
A SIMPLE GENE COMPRISING TWO EXONS
E1 E25’3’
5’ 3’
PRINCIPLES OF PCR AMPLIFICATION
3’ 5’
5’ 3’
5’ 3’
3’ 5’
5’ 3’
5’ 3’
5’ 3’
3’ 5’
3’ 5’
FIRST ROUND
SECOND ROUND
Exponential increase of the number of copies during PCR
PCR AMPLIFICATION
M13-FOR TGTAAAACGACGGCCAGTM13-R EV CAGGAAACAGCTATGACC
SIMPLICITY OF PRIMER DESIGN
E1 E2
Annealing TmMgCl2
GC Buffer
58°C1.5mM
+
60°C, 2.5mM
EVOLUTION OF CHAOS
ONE GENE MANY GENES
Five annealing temperaturesFour Mg concentrationsEach amplicon sequenced using two gene-specific oligos
56°C, 2mM 57.5°C, 1.5mM
55°C, 1mM 62°C, 1mM
Tm 60°C2.0mM MgCl2+/-GC Buffer
Fast Start Taq DNA Polymerase
M13 tails for all primers
AIM: TO ACHIEVE SIMPLICITY IN PRIMER DESIGN AND AMPLIFICATION
EXON-SPECIFIC PRIMER PAIR
chr16:67406743+67407333 591bp TTGAGAAGCCATGGTAAGTAATTG GAAGGGAACAGGTGAAAGGAGTTGAGAAGCCATGGTAAGTAATTGtggtttctgccattgaaagtcatggcagaaaccacagttacttttgcaccaacctaatatattaccaaaagcaacagttaaggatttaattttatttttactaacacaaaatgtttcgttttgtttttaacttcattgtttctgctctctagggcttggattttgaggccaagcagcagtacattctacacgtagcagtgacgaatgtggtaccttttgaggtctctctcaccacctccacagccaccgtcaccgtggatgtgctggatgtgaatgaagcccccatctttgtgcctcctgaaaagagagtggaagtgtccgaggactttggcgtgggccaggaaatcacatcctacactgcccaggagccagacacatttatggaacagaaaataacgtaagtgtgaggatttttcaactgacttgcagcaactggttattttatatcattttatatgtaaatcaataatatgtacttcatggcattttgtcatttgtctgtacaagaccattctcttaatttattttttattccctttatctgtgCTCCTTTCACCTGTTCCCTTC
Forward: 59.6 C ttgagaagccatggtaagtaattgReverse: 60.1 C gaagggaacaggtgaaaggagThe temperature calculations are done assuming 50 mM salt and 50 nM annealing oligoconcentration.
EXON20bp 20bp
Splice acceptor site Splice donor site
AMPLICONS: LQT GENE EXONS IN PLATE FORMAT
CODING SEQUENCE OF LQT GENES 1, 2, 3, 5, 6 AND 7
96 well384 well
CURRENTLY USE CAPILLARY-BASED SEQUENCING
LIQUID HANDLING ROBOT
AMPLICON PURIFICATION (Exosap; Ampure)
SEQUENCE SET-UP
SEQUENCE CLEAN-UP (Cleanseq)
LABORATORY AUTOMATION
SEQUENCE ANALYSIS SOFTWARE
SeqMan (DNASTAR)Sequencher™ (GENE CODES CORPORATION)Mutation Surveyor®/Explorer® (SOFTGENETICS)StadenSeqScape® (APPLIED BIOSYSTEMS)
Variant Reporter™ (APPLIED BIOSYSTEMS)
IMPORT GENBANK SEQUENCE WITH ANNOTATIONSor
IMPORT YOUR OWN REFERENCE SEQUENCE AND ANNOTATE MANUALLY
GENE CONNECTION FOR THE HEARThttp://www.fsm.it/cardmoc/
THE FUTURE IS NOW
THINK DIFFERENTLY.
JUST DO IT!
NEXT GENERATION: PARALLEL (DEEP) SEQUENCING
PCR AMPLIFY REGIONS OF INTEREST
POOL PRODUCTS
SEQUENCE(BY SYNTHESIS)
HIGH THROUGHPUT PARALLEL SEQUENCING:ROCHE GS-FLX SEQUENCING PLATFORM
HIGH THROUGHPUT PARALLEL SEQUENCINGROCHE GS-FLX SEQUENCING PLATFORM
ABILITY TO POOL PATIENTS’ AMPLICONS
REQUIRES DIFFERENTIALLY TAGGED(BAR CODED)
PRIMERS FOR EACH PATIENT
HIGHER LEVEL POOLING CONCEPT
PARALLEL SEQUENCING APPROACH
PCR AMPLIFY REGIONS OF INTEREST
POOL PRODUCTS
SEQUENCE
FRAGMENT GENOMIC DNA
ADD LINKERS
CAPTURE DEFINED AMPLICONS
ELUTE CAPTURED AMPLICONS
SEQUENCE
AGILENT SURE-SELECTARRAY-BASED CAPTURE
GS-FLX SEQUENCING
Albert et al, Nature Methods 2007;4:903-905
ARRAY-BASED CAPTURE
GS-FLX SEQUENCING
DON LOVE DIAGNOSTIC GENETICS
FISHKARYOTYPING
SEQUENCING
PROTEIN MODELLING
QUANTITATION
CNV
MODERN TRENDS IN GENOME ANALYSIS:CAN WE DO IT, SHOULD WE DO IT?
1
2
ARRAY CGH
AFFYMETRIX SNP 6.0 CHIPS
CHARACTERISATION OF CHROMOSOME 10 INTERSTITIAL DELETION
ARRAY CGH : 46,XX,del(10)(q23.1q23.2)
ALTERNATIVE ANALYSIS OF SNP DATA USING RECENTLY RELEASED AFFYMETRIX SOFTWARE
ARRAY CGH : 46,XX,del(10)(q23.1q23.2)
IDENTIFICATION OF GENES THAT
LIE IN THE DELETED REGION
The deletion resolved to 81,619,836bp-89,084,996bp on chromosome 10Multiple genes are located within this region
Susceptibility to severe respiratory syncytial virus infection
Brain demyelination through MAT1A deficiency
Susceptibility to schizophrenia
Predisposition to juvenile polyposis
ARRAY CGH : 46,XX,del(10)(q23.1q23.2)
ARRAY CGH: NEW WORK FLOWGENOMIC DNA
WHOLE GENOME AMPLIFICATION100ng, 30ºC, 3 HOURS
ENZYMATIC CLEAVAGEAND LABELLING OF FRAGMENTS
HYBRIDIZATION TO CHIPWASH CHIPSCAN CHIP
THURSDAY
FRIDAY
THE FOCUS IS ON TRISOMY 21
NON-INVASIVE PRENATAL DIAGNOSIS (NIPD)
SEQUENOM CORE TECHNOLOGY IS BASED ON SEQUENOM CORE TECHNOLOGY IS BASED ON MALDIMALDI--TOF MSTOF MS
1
+
+
+
+
• Samples spotted on chip• Chip is placed in machine• Machine is vacuum tube with electric field with
detector at end of tube
• Laser “zaps” sample• Laser tuned such that molecules get same
charge
• Molecules “zapped” by the laser become charged
• Samples accelerate in electrical field• Smaller molecules accelerate faster
• Molecules hit detector• Order of peak indicates size of molecule• Area of peak indicates abundance
1
12
13
14
15
+
+
+
6600 6700 6800 6900
UEP.M
MBT01167CC
T
CTT
UEP.M
MBT05641 CC
T
CTT
UEP.M
MBT05728
UEP.M
MBT10830
MMBT05728
Mass
A T G T
10 mer tag
10 mer tag
10 mer tag
10 mer tag
A TT A
G TC A
A G
Primer ExtensionA,C,T,G terminators
PCR
Sample ConditioningSpot on SpectroCHIPFly on Mass Spec
SAPHybridize Extension Primer
OVERVIEW OF GENOTYPING ASSAYOVERVIEW OF GENOTYPING ASSAY
Allele 1 Allele 2
A GA
normal fetus trisomy 21 fetus
SNP on PLAC4(chromosome 21)
transcription
PLAC4 RNAexpressed in
placenta
deviation in RNA-SNP
allelic ratio
release into maternal circulation
circulating PLAC4RNA in maternal
plasma
deviation in RNA-SNP
allelic ratio
GA
RNA-SNP ALLELIC RATIO
PCR amplification
Relative peak area: 0.5 0.5 0.67 0.33
Allelic ratio = allele-G peak areaallele-A peak area
1 0.5
Base extension
Mass detection
Mass
Inte
nsity
allele-Aallele-G
Mass
Inte
nsity
allele-Aallele-G
RNA cDNA
normal fetus trisomy 21 fetus
RNARNA--SNP ALLELIC RATIOSNP ALLELIC RATIO
CHALLENGESCHALLENGES
BIOLOGICAL RELEVANCECLINICAL UTILITY
INFRASTRUCTURE DEVELOPMENTIMPLEMENTATION
ACKNOWLEDGEMENTSElaine Doherty
Anne Vaughan, Debbie Prosser, Jenny LoveAnna Zhang, Stella Lai
Christina Lim, Jamie-Lee Day