Transcript Chapter 21: Molecular Basis of Cancer

Molecular diagnosis of heterogeneous genetic
diseases: the example of muscular dystrophies
Vincenzo Nigro
Dipartimento di Patologia
Generale, Seconda Università
degli Studi di Napoli
Telethon Institute of Genetics and
Medicine (TIGEM)
What is a mutation?
A variation of the DNA sequence
 that is only found in affected individuals
 that is never found in non affected
individuals
 that accounts for the pathological
process/status
 that, when corrected in time, disease is
rescued
..that is only found in affected and
that is never found in non affected
incomplete penetrance
that is more often found in affected
than in non affected...
50.000 private variants = innocuous
differences belonging to one family
CCCCAGCCTCCTTGCCAACGCCCCCTTTCCCTCTCCCCCTCCCGCTCGGCGCTGACC
CCCCATCCCCACCCCCGTGGGAACACTGGGAGCCTGCACTCCACAGACCCTCTCCTT
GCCTCTTCCCTCACCTCAGCCTCCGCTCCCCGCCCTCTTCCCGGCCCAGGGCGCCG
GCCCACCCTTCCCTCCGCCGCCCCCCGGCCGCGGGGAGGACATGGCCGCGCACAG
GCCGGTGGAATGGGTCCAGGCCGTGGTCAGCCGCTTCGACGAGCAGCTTCCAATAA
AAACAGGACAGCAGAACACACATACCAAAGTCAGTACTGAGCACAACAAGGAATGTC
TAATCAATATTTCCAAATACAAGTTTTCTTTGGTTATAAGCGGCCTCACTACTATTTTAA
AGAATGTTAACAATATGAGAATATTTGGAGAAGCTGCTGAAAAAAATTTATATCTCTCT
CAGTTGATTATATTGGATACACTGGAAAAATGTCTTGCTGGGCAACCAAAGGACACAA
TGAGATTAGATGAAACGATGCTGGTCAAACAGTTGCTGCCAGAAATCTGCCATTTTCT
TCACACCTGTCGTGAAGGAAACCAGCATGCAGCTGAACTTCGGAATTCTGCCTCTGG
GGTTTTATTTTCTCTCAGCTGCAACAACTTCAATGCAGTCTTTAGTCGCATTTCTACCA
GGTTACAGGAATTAACTGTTTGTTCAGAAGACAATGTTGATGTTCATGATATAGAATTG
TTACAGTATATCAATGTGGATTGTGCAAAATTAAAACGACTCCTGAAGGAAACAGCAT
TTAAATTTAAAGCCCTAAAGAAGGTTGCGCAGTTAGCAGTTATAAATAGCCTGGAAAA
GGCATTTTGGAACTGGGTAGAAAATTATCCAGATGAATTTACAAAACTGTACCAGATC
CCACAGACTGATATGGCTGAATGTGCAGAAAAGCTATTTGACTTGGTGGATGGTTTTG
CTGAAAGCACCAAACGTAAAGCAGCAGTTTGGCCACTACAAATCATTCTCCTTATCTT
GTGTCCAGAAATAATCCAGGATATATCCAAAGACGTGGTTGATGAAAACAACATGAAT
AAGAAGTTATTTCTGGACAGTCTACGAAAAGCTCTTGCTGGCCATGGAGGAAGTAGG
CAGCTGACAGAAAGTGCTGCAATTGCCTGTGTCAAACTGTGTAAAGCAAGTACTTACA
TCAATTGGGAAGATAACTCTGTCATTTTCCTACTTGTTCAGTCCATGGTGGTTGATCTT
AAGAACCTGCTTTTTAATCCAAGTAAGCCATTCTCAAGAGGCAGTCAGCCTGCAGATG
TGGATCTAATGATTGACTGCCTTGTTTCTTGCTTTCGTATAAGCCCTCACAACAACCAA
CACTTTAAGATCTGCCTGGCTCAGAATTCACCTTCTACATTTCACTATGTGCTGGTAAA
TTCACTCCATCGAATCATCACCAATTCCGCATTGGATTGGTGGCCTAAGATTGATGCT
GTGTATTGTCACTCGGTTGAACTTCGAAATATGTTTGGTGAAACACTTCATAAAGCAG
TGCAAGGTTGTGGAGCACACCCAGCAATACGAATGGCACCGAGTCTTACATTTAAAG
AAAAAGTAACAAGCCTTAAATTTAAAGAAAAACCTACAGACCTGGAGACAAGAAGCTA
TAAGTATCTTCTCTTGTCCATGGTGAAACTAATTCATGCAGATCCAAAGCTCTTGCTTT
GTAATCCAAGAAAACAGGGGCCCGAAACCCAAGGCAGTACAGCAGAATTAATTACAG
GGCTCGTCCAACTGGTCCCTCAGTCACACATGCCAGAGATTGCTCAGGAAGCAATGG
AGGCTCTGCTGGTTCTTCATCAGTTAGATAGCATTGATTTGTGGAATCCTGATGCTCC
TGTAGAAACATTTTGGGAGATTAGCTCACAAATGCTTTTTTACATCTGCAAGAAATTAA
CTAGTCATCAAATGCTTAGTAGCACAGAAATTCTCAAGTGGTTGCGGGAAATATTGAT
CTGCAGGAATAAATTTCTTCTTAAAAATAAGCAGGCAGATAGAAGTTCCTGTCACTTTC
CCCCAGCCTCCTTGCCAACGCCCCCTTTCCCTCTCCCCCTCCCGCTCGGCGCTGACC
CCCCATCCCCACCCCCGTGGGAACACTGGGAGCCTGCACTCCACAGACCCTCTCCTT
GCCTCTTCCCTCACCTCAGCCTCCGCTCCCCGCCCTCTTCCCGGCCCAGGGCGCCG
GCCCACCCTTCCCTCCGCCGCCCCCCGGCCGCGGGGAGGACATGGCCGCGCACAG
GCCGGTGGAATGGGTCCAGGCCGTGGTCAGCCGCTTCGACGAGCAGCTTCCAATAA
AAACAGGACAGCAGAACACACATACCAAAGTCAGTACTGAGCACAACAAGGAATGTC
TAATCAATATTTCCAAATACAAGTTTTCTTTGGTTATAAGCGGCCTCACTACTATTTTAA
AGAATGTTAACTATATGAGAATATTTGGAGAAGCTGCTGAAAAAAATTTATATCTCTCT
CAGTTGATTATATTGGATACACTGGAAAAATGTCTTGCTGGGCAACCAAAGGACACAA
TGAGATTAGATGAAACGATGCTGGTCAAACAGTTGCTGCCAGAAATCTGCCATTTTCT
TCACACCTGTCGTGAAGGAAACCAGCATGCAGCTGAACTTCGGAATTCTGCCTCTGG
GGTTTTATTTTCTCTCAGCTGCAACAACTTCAATGCAGTCTTTAGTCGCATTTCTACCA
GGTTACAGGAATTAACTGTTTGTTCAGAAGACAATGTTGATGTTCATGATATAGAATTG
TTACAGTATATCAATGTGGATTGTGCAAAATTAAAACGACTCCTGAAGGAAACAGCAT
TTAAATTTAAAGCCCTAAAGAAGGTTGCGCAGTTAGCAGTTATAAATAGCCTGGAAAA
GGCATTTTGGAACTGGGTAGAAAATTATCCAGATGAATTTACAAAACTGTACCAGATC
CCACAGACTGATATGGCTGAATGTGCAGAAAAGCTATTTGACTTGGTGGATGGTTTTG
CTGAAAGCACCAAACGTAAAGCAGCAGTTTGGCCACTACAAATCATTCTCCTTATCTT
GTGTCCAGAAATAATCCAGGATATATCCAAAGACGTGGTTGATGAAAACAACATGAAT
AAGAAGTTATTTCTGGACAGTCTACGAAAAGCTCTTGCTGGCCATGGAGGAAGTAGG
CAGCTGACAGAAAGTGCTGCAATTGCCTGTGTCAAACTGTGTAAAGCAAGTACTTACA
TCAATTGGGAAGATAACTCTGTCATTTTCCTACTTGTTCAGTCCATGGTGGTTGATCTT
AAGAACCTGCTTTTTAATCCAAGTAAGCCATTCTCAAGAGGCAGTCAGCCTGCAGATG
TGGATCTAATGATTGACTGCCTTGTTTCTTGCTTTCGTATAAGCCCTCACAACAACCAA
CACTTTAAGATCTGCCTGGCTCAGAATTCACCTTCTACATTTCACTATGTGCTGGTAAA
TTCACTCCATCGAATCATCACCAATTCCGCATTGGATTGGTGGCCTAAGATTGATGCT
GTGTATTGTCACTCGGTTGAACTTCGAAATATGTTTGGTGAAACACTTCATAAAGCAG
TGCAAGGTTGTGGAGCACACCCAGCAATACGAATGGCACCGAGTCTTACATTTAAAG
AAAAAGTAACAAGCCTTAAATTTAAAGAAAAACCTACAGACCTGGAGACAAGAAGCTA
TAAGTATCTTCTCTTGTCCATGGTGAAACTAATTCATGCAGCTCCAAAGCTCTTGCTTT
GTAATCCAAGAAAACAGGGGCCCGAAACCCAAGGCAGTACAGCAGAATTAATTACAG
GGCTCGTCCAACTGGTCCCTCAGTCACACATGCCAGAGATTGCTCAGGAAGCAATGG
AGGCTCTGCTGGTTCTTCATCAGTTAGATAGCATTGATTTGTGGAATCCTGATGCTCC
TGTAGAAACATTTTGGGAGATTAGCTCACAAATGCTTTTTTACATCTGCAAGAAATTAA
CTAGTCATCAAATGCTTAGTAGCACAGAAATTCTCAAGTGGTTGCGGGAAATATTGAT
CTGCAGGAATAAATTTCTTCTTAAAAATAAGCAGGCAGATAGAAGTTCCTGTCACTTTC
1-allele diseases
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monoallelic mutations may be
responsible for dominant or X-linked
disorders
new random mutations are the rule
with an unpredictable pattern of
distribution
Gender effect in mutations
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For mutations other than point mutations, sex
biases in the mutation rate are very variable
Small deletions are more frequent in females
Germline base substitution mutations occur more
frequently in males than in females, especially in
older males
Point mutations at some loci occur almost
exclusively in males, whereas others occur ten
times more than in females
Relative frequency of de novo achondroplasia for
different paternal ages
Relative frequency of de novo
neurofibromatosis for different paternal ages
the number of male germ-cell
divisions
2-allele diseases
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novel mutations are rare, usually mutations
have a long history (100-1000 generations)
mutations have an ethnical signature with a
predictable pattern of distribution and
frequency
biallelic mutations may be responsible for
autosomal recessive disorders
polymorphisms and private variants are more
easily discriminated vs true mutations
2-allele diseases
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consanguineity is a risk factor for
homozygosity
high carrier frequency is a risk factor for
compound heterozygosity
The effect of an allele

null or amorph = no product

hypomorph = reduced amount / activity

hypermorph = increased amount / activity

neomorph = novel product / activity
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antimorph = antagonistic product / activity
Dominant or recessive phenotype?
Loss of function mutations in the PAX3
gene (Waardenburg syndrome)
haploinsufficiency
amorph / hypomorph (1)
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deletion
– the entire gene
– part of the gene
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disruption of the gene structure
– by insertion, inversion, translocation
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promoter inactivation
mRNA destabilization
splicing mutation
– inactivating donor/acceptor
– activating criptic splice sites
amorph / hypomorph (2)
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frame-shift in translation
– by insertion of n+1 or n+2 bases into the
coding sequence
– by deletion of n+1 or n+2 bases into the
coding sequence
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nonsense mutation
missense mutation / aa deletion
– essential / conserved amino acid
– defect in post-transcriptional processing
– defect in cellular localization
hypermorph
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trisomia
duplication
amplification (cancer)
chromatin derepression (FSH)
trasposition under a strong promoter
– leukemia

overactivity of an abnormal protein
neomorph
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generation of chimeric proteins
duplication
amplification (cancer)
missense mutations
inclusion of coding cryptic exons
usage of alternative ORFs
overactivity of an abnormal protein
antimorph
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missense mutations
inclusion of coding cryptic exons
usage of alternative ORFs
Mutation detection
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mutation scanning
– or resequencing methods for identifying
previously unknown mutations

genotyping
– methods for scoring previously known
mutations or single nucleotide
polymorphisms (SNPs)
Key questions for mutation
detection strategy
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expected mutations are monoallelic or biallelic?
is the gene well recognized for that disease?
is the mutation pattern known? (deletion, dup,
small mutations, etc.)
which is the complexity of the gene?
how many patients must be examined?
how many controls should be examined?
how many mutations and how many variations
have already been identified in this gene?
are there more members of the same gene family
(or pseudogenes) in the genome?
Dimension of the mutation detection study
Number of
patients
Gene size
X
Number of
controls
General strategy for mutation detection
screening
of recurrent
mutations
frequent
mutations
are known?
YES
mutations
are identified?
NO
NO
YES
SEQUENCING
mutation
scanning

DMD Duchenne Muscular Dystrophy
- 1/3,500 boys
Onset -- Early childhood - about 2 to 6 years
– Laboratory -- CK (50x to 1.000x), LDH5, ALT,
AST, aldolase increase
Symptoms -- Generalized weakness and muscle
wasting affecting proximal limb muscles first.
Calves often enlarged. Heart involvement
Progression -- Disease progresses slowly but will
affect all voluntary muscles. Survival possible
beyond late twenties

BMD Becker Muscular Dystrophy
- 1/10,000 boys
Onset -- Adolescence or adulthood
Symptoms -- Almost identical to Duchenne but
often much less severe. Heart involvement
Progression -- Slower and more variable than
DMD with survival well into mid to late adulthood
Carrier of a balanced reciprocal X-autosome
translocation
Dystrophin gene: page 1/185
Dystrophin gene: page 2/185
Dystrophin gene: page 3/185
Dystrophin gene: page 185/185
Telethon-UILDM
250/300
DMD/BMD
Qualitative test
rejected
Quantitative test
more DNA
80plex-PCR
Deletions
duplications
Point mutations
mRNA study
Family tests
Log-PCR = 4 multiplex-PCR (2x20+2x18) with uniform spacing
and gel position according to chromosomal position
A
DMD patient :
groups A, B
BMD patient :
groups C, D
Deletion ex 17-43
Duplication ex 13-23
C
B
DMD
BMD
1: del ex 43
2: del ex 11, 17, 19, 21
3: del ex 17, 19, 21
4: del ex 50, 52
5: del ex 7, 11, 17, 19
6: del ex 61
1
2
3
4
5
6
1: no del
2: del ex 8, 12, 18, 20, 22
3: del ex 12, 18, 20, 22
4: del ex 46, 51
5: del ex 6, 8, 12, 18
6: del ex 62
D
large deletions in 377/506 DMD/BMD
74.5%
large duplications in 51/506 patients
10.1%
SALSA MLPA probes
Hybridysation
1.
2.
The MLPA probemix is added to denatured
genomic DNA
The two parts of each probe hybridise to adjacent
target sequences
Ligation
3.
Probes are ligated by a thermostable ligase
PCR amplification
4.
A universal primer pair is used to amplify all
ligated probes
The PCR product of each probe has a unique
length (130 480 bp)
Separation and quantification by
capillary electrophoresis
Each peak is the
amplification product
of a specific probe.
Samples are
compared to a control
sample.
A difference in relative
peak height or peak
area indicates a copy
number change of the
probe target sequence
Detection of Chr X copy number
X
Male
Female
Triple X
283 bp
346 bp
MRC-Holland b.v.
MLPA discriminates sequences that differ in only a single nucleotide
and can be used to detect known mutations.
Mismatch
Mismatch at the probe ligation site 
No ligation, no amplification product
Perfect match
Ligation of the two probe oligonucleotides
 Amplification product
MS-MLPA
M
M
Methylated Target
Denaturation and
Multiplex probe
hybridization
Ligation and
Digestion with
methylation
sensitive
endonucleases
Unmethylated Target
M
M
Only undigested (methylated) and ligated probes are exponentially amplified
MRC-Holland b.v.
Limb-girdle weakness
proximal weakness: most common
 Lower extremities
– difficulty climbing stairs
– arising from a low chair or toilet
– getting up from a squatted position
 Upper extremities
– trouble lifting objects over their head
– brushing their hair
 distal weakness
– difficulty opening jars, inability to turn a key in
the ignition, or tripping due to foot drop
 cranial weakness
– dysarthria, dysphagia or ptosis
Genetics of limb-girdle muscular dystrophies
autosomal dominant
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LGMD1A
LGMD1B
LGMD1C
LGMD1D
LGMD1E
LGMD1F
LGMD1G
5q31.2
1q21
3p25.3
6q22
7q35
7q31.1
4p21
autosomal recessive
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LGMD2A
LGMD2B
LGMD2C
LGMD2D
LGMD2E
LGMD2F
LGMD2G
LGMD2H
LGMD2I
LGMD2J
LGMD2K
LGMD2L
LGMD2M
15q15
2p13.2
13q12
17q21.33
4q12
5q33
17q12
9q33 .1
19q13.3
2q24.3
9q34.1
9q31
11p13-p12
myotilin (Hauser, 2000)
lamin A/C (Bonne, 1999)
caveolin 3 (Minetti, 1997)
?
?
filamin C
?
calpain 3 (Richard, 1995)
dysferlin (Bashir, Liu, 1998)
g-sarcoglycan (Noguchi, 1995)
a-sarcoglycan (Roberds, 1994)
b-sarcoglycan (Bonnemann, Lim, 1995)
d-sarcoglycan (Nigro, 1996)
telethonin (Moreira, 2000)
TRIM 32 (Frosk, 2002)
FKRP (Brockington, 2001)
titin (Udd, 2002)
POMT1 (Balci, 2005)
fukutin (Godfrey, 2006)
?
autosomal dominant
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autosomal dominant forms (LGMD1)
are generally milder
represent less than 10% of all LGMD
marked heterogeneity for LGMD1, one
gene per one single family
autosomal recessive
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autosomal recessive forms (LGMD2) have an
average prevalence of 1:14,000-1:20,000 at birth
frequency differences among countries
this depends on higher carrier frequencies of single
mutations, as 550delA for calpain 3 in Croatia,
L276I for FKRP in Northern Europe, 521delT for
gamma-sarcoglycan in Northern Africa
At least 25% of families are excluded from any
known locus and 40% of typical LGMD cases have
no mutation in any known gene
Tools to address the
diagnosis of LGMD
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Clinical presentation (MRI)
WB analysis
Segregation study
Mutation detection in patients
Mutation detection in normal subjects
Homogeneous collection of mutations
and polymorphisms
Segregation analysis
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Analysis of 30 polymorphic markers linked to
LGMD2A, 2B, 2C-2F, 2I in sib pairs
To find homozigosity…
Calpain 3
24 exons
Myotilin
dysferlin
55 exons
Lamin A/C
13 exons
a-sarcoglycan 10 exons
Caveolin 3
2 exons (3)
b-sarcoglycan 6 ex (7)
g-sarcoglycan 8 ex (10)
d-sarcoglyican 9 exons
Telethonin
2 exons (3)
TRIM32
1 exons (7)
FKRP
4 esons (8)
Titin
363 ex (35)
9 exons
Case 1
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The gene is known
It is composed of five small size exons
There are 10 patients, sons of consanguineous parents
Expected mutations are homozygous
Mutations have never been identified in this gene
There is no other member of the same gene families
(or pseudogenes) in the genome
Case 2
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The gene is known
The putative function of the gene product is to
serve as a transcription factor
Expected mutations are dominant
Mutations have never been identified in this
gene
There are other members of the same gene
families (or pseudogenes) in the genome
Sequencing
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With the ongoing reduction of costs (today
about 2-4 €/run), sequencing of PCR products
is applied for mutation detection
Sequencing is often thought of as the 'gold
standard' for mutation detection.
This perception is distorted due to the fact
that this is the only method of mutation
identification, but this does not mean it is the
best for mutation detection
Sequencing artifacts
FALSE POSITIVE (specificity)
–when searching for heterozygous DNA differences
there are a number of potential mutations, together
with sequence artifacts, compressions and
differences in peak intensities that must be rechecked with additional primers and costs
FALSE NEGATIVE (sensitivity)
–loss of information farther away or closer to the
primer
–does not detect a minority of mutant molecules in a
wild-type environment
Current mutation scanning
techniques
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SSCP (single strand conformation
polymorphism)
HA (heteroduplex analysis)
CCM (chemical cleavage of mismatch)
CSGE (conformation sensitive gel
electrophoresis)
DGGE (denaturing gradient gel
electrophoresis)
DHPLC (denaturing HPLC)
PTT (protein truncation test)
DGCE (denaturing gradient capillary
electrophoresis)
direct sequencing
SSCP
Mutation detection by
heteroduplex analysis:
the mutant DNA must
first be hybridized with
the wild-type DNA
to form a mixture of two
homoduplexes and two
heteroduplexes
Heteroduplex analysis
DHPLC
denaturing HPLC
from Transgenomic
DHPLC analysis
at different
temperatures of
the column
Analysis of dystrophin exon 59
3.88
0.49
2.5
Homoduplex
DNA:
PCR fragments
are identicals
6.41
2.0
Intensity (mV)
1.5
1.0
0.5
0.0
0
1
2
3
4
5
Time
6
8
6.39
Heteroduplex
DNA:
PCR fragments
are different
3.65
3.85
1.5
Intensity (mV)
7
(min)
0.49
Retention
1.0
0.5
0.0
0
1
2
3
4
Retention
5
Time
(min)
6
7
8
DHPLC analysis of the CAPN3 gene (exon
11)
UV
2
0
1:2
FLUO
100
0
1:4
1:6
1:8
1:10
PLATE B
PLATE A
POOLED PLATES
A+B
DHPLC analysis
c.3285_3288 del CAGT
c.2880_2884 del CAAAC
stop
splicing
c.3336 del G
c.4100 delA
frame-shift
c.2302 C>T R768X
missense
c.4326 delG
Q1564X
c.2125 C>T Q709X
R1577X
c.1482 delG
c.3464_3471 del GTTTGGAG
c.1332-9 A>G
R3370X
c.5091del G
c.1292 G>A W431X
c.9926_9929 ins AAGC
S805X
c.1300_1310 del CTCAGGGTAGC
c.6353 delA
c.1180 del G
c.8732 insA
c.5690 ins A
c.713_714 delTT
C3337Y
E1925X
c.530+1 G>A
c.9429_9430 del GC
c.583 C>T R195X
c.8391-2 A>G
R1967X
c.94-1G>A
S2008X
c.401_404 del CCAA
Q242X
Q1737X
S622X
c.6980 delA
E3277X
c.7006 C>T Q2336X
Y3158X
Q986X
R1314X
c.433 C>T R145X
c.1062 G>A W354X
Q1087X
K105X
c.1390 del C
c.1603 delGTAinsCT
9563+1 A>G
c.4119 del G
c.8668+3 A>T
R1844X
R2982X
c.4871_4872 del AG
R1666X
Q1373X
R2905X
c.10223+1 G>A
c.9204_9207 del CAAA
3367 del E
PTT
protein truncation test
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Sensitivity
1000-bp fragment > 85%
Detects only nonsense mutations
Post PCR time: 48-72 hours
(translation/trascription, gel preparation,
loading and run, analysis of results)
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Use of 35S radioactivity
No special equipment required
mRNA as starting template
Applications of PTT
(% of truncating mutations)
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Polycystic Kidney Disease PKD1
95%
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Familial Adenomatous Polyposis APC
95%
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Ataxia telangiectasia ATM
90%
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Hereditary breast and ovarian cancer BRCA1-2
90%
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Duchenne Muscular Dystrophy DMD
90%?
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Fanconi anemia FAA
80%
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Hereditary non-polyposis colorectal cancer hMSH1-2 70%80%
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Neurofibromatosis type 2 NF2
65%
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Hunter Syndrome IDS
50%
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Neurofibromatosis type 1 NF1
50%
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Cystic Fibrosis CFTR
15%
Molecular inversion probe (MIP) genotyping
•MIP genotyping uses circularizable probes with 5′ and
3′ ends that anneal upstream and downstream of the
SNP site leaving a 1 bp gap
•Polymerase extension with dNTPs and a non-stranddisplacing polymerase is used to fill in the gap
•Ligation seals the nick, and exonuclease I is used to
remove excess unannealed and unligated circular probes
•The resultant product is PCR-amplified and the
orientation of the primers ensures that only circularized
probes will be amplified
•The resultant product is hybridized and read out on an
array of universal-capture probes
GoldenGate genotyping assay
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GoldenGate uses extension ligation between
annealed locus-specific oligos (LSOs) and allelespecific oligos (ASOs)
An allele-specific primer extension step is used to
preferentially extend the correctly matched ASO (at
the 3′ end) up to the 5′ end of the LSO primer
Ligation then closes the nick
GoldenGate genotyping assay
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A subsequent PCR amplification step is used to amplify
the appropriate product using common primers to
‘built-in’ universal PCR sites in the ASO and LSO
sequences
The resultant PCR products are hybridized and read
out on an array of universal-capture probes
454 technology:
DNA fragmentation and adaptor ligation
454 technology:
a water-in-oil emulsion is created:
a single molecule of DNA with a single bead
454 technology:
Beads with clones are selected and
assembled onto a planar substrate
454 technology:
Sequencing by synthesis
pyrosequencing
Up to 100 Million bp
in 8 hours can be
read
Ambiguities arise for
homopolymeric
tracts
7.4 x coverage
234 runs
24.5 billions bp
11 genetic diseases !!
NimbleGen sequence capture