Transcript Document

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Methods for detection of
un known mutations
BRCA
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BRCA1 Gene
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BRCA2 Gene
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SSCP
single strand conformation
polymorphism
simplicity
clearly by heteroduplex analysis (HA)
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SSCP
SSCP Gels
Prepare 0.5x MDE gel as follows:
MDE gel16.0mlddH2O44.2ml10X
TBE3.84ml10% APS256µlTEMED25.6µlPour
sequencing gel format with appropriate
sharkstooth comb. Gel will polymerize in
about 1 hour
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SSCP
Loading Buffer
95% formamide
10mM NaOH
0.025% Bromophenol Blue
0.025% Xylene Cyanol
Run gel in 0.6X TEB buffer.
Heat denature samples at 94°C for
5 minutes and place them on ice
for 3-5 minutes. Load 2.0-4.0µl per
sample. Include non-denatured
controls
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Electrophoresis conditions
Fragment Size: 150-200 bp
6 Watts
10-12 hours
room temperature
Fragment Size: > 200 bp
8 Watts
10-12 hours
room temperature
Exposure
Dry gel and expose either at -80°C for 2
hours or at room temperature for 16-18
hours.
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Pedigree of a selected family with
breast cancer
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SSCP Analysis
BRCA1 Exon 15, 4650delCA
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Pedigree of a selected family
with breast cancer
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SSCP
Analysis
BRCA1, Exon
20,
Nt 5382
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SSCP Analysis
Exon 11pi BRCA1 MS R1347G
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Protein truncation test
PTT
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PTT
• For BRCA1/2 using the Protein
Truncation Test (PTT) for exon 11 of
BRCA1 & exon 10-11 of BRCA2
• These exons cover approximately
over 60% of each gene
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PTT
Coding sequence without
introns
cDNA via RT-PCR from RNA
or large exons in genomic
DNA
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cDNA
It is PCR amplified
The forward primer carries at its 5'
end a T7 promoter
followed by a eukaryotic translation
initiation sequence
which includes an ATG start codon
Next is a gene-specific sequence
designed so that the sequence
amplified reads in-frame from the
ATG
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Protein truncation test (PTT)
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PTT
 After amplification
 the PCR product is added to a coupled in
vitro transcription-translation system
 For detection a labelled amino acid is
included
 which is usually methionine, leucine or
cysteine
 The label can either be a radionucleotide
such as [35S]
which is visualised by autoradiography
 Or biotin which is detected by a colorimetric
Western blot employing a streptavidin-biotinalkaline phosphatase complex
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PTT
The polypeptides produced are separated
by size using an SDS-PAGE gel.
If the product is only full length
no truncating mutation is present
Truncating mutations result in shorter
products
the size of which gives the approximate
position of the mutation.
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Protein truncation test
used in diagnostic laboratories
dealing with cancer genes
because they often contain
truncating mutations.
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Protein truncation test (PTT)
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A nonisotopic protein
truncation test
• WT is wild-type DNA
• C1−C3 are mutant
homozygous DNA
samples from cell lines
• P1−P4 are the
heterozygous DNA
samples from patients
diagnosed with FAP
• BL1/2: a cell-free
translation performed
lacking both tRNAs
and DNA
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The protein truncation test (PTT)
 First, RNA is reverse transcribed (RT) to generate a cDNA
copy.
 Second, the cDNA (or genomic DNA) is amplified using
the polymerase chain reaction (PCR) in combination
with a specifically tailed forward primer facilitating in
vitro transcription by T7-RNA polymerase.
 Products are analyzed on agarose gel to verify
amplification
 abnormally migrating products point to mutations
Deletions
Duplications
affecting splicing
 Finally, in vitro transcription/translation is used to
generate peptide fragments
analyzed on SDS-PAGE gel
to detect translation terminating mutations
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The protein truncation test (PTT)
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ADVANTAGES
Detects truncating mutations
Allows the analysis of large stretches of
coding sequence (up to 5 kb: 2kb:genomic
DNA, 1.3-1.6kb cDNA is best)
Either: large single exons (DNA template)
or multiple exons (RNA template).
Length of the truncated protein pinpoints
the position of the mutation, thereby
facilitating its confirmation by sequencing
analysis
SENSITIVITY: the sensitivity of PTT is good
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DISADVANTAGES
Not applicable to all genes
E.g. APC, BRCA1, BRCA2 and
Dystrophin all have approximately 9095% truncating mutations
but NF1 has only 50% truncating
mutations respectively
Most powerful as a technique when
RNA is used, however, most
laboratories only have DNA stored.
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DISADVANTAGES
The most readily available source of RNA is
blood.
However expression of the target gene in this
tissue may be low, requiring technically more
demanding nested amplification reactions to
obtain sufficient signal.
Cannot detect mutations occurring outside the
coding region, which affect control of
expression and RNA stability
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Deletions/insertions/duplications
•Out of frame
•In frame
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Deletions/insertions/duplications
Out of frame:
result in frameshifts giving rise to
stop codons.
no protein product or truncated
protein product
 deletions/insertions in DMD
patients : truncated dystrophins of
decreased stability
RB1 gene - usually no protein
product in retinoblastoma
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Deletions/insertions/duplications
In frame:
loss or gain of amino acid(s)
depending on the size and may give
rise to altered protein product with
changed properties
eg CF Delta F508 loss of single amino
acid
In some genes loss or gain of a single
amino acid: mild
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In frame:
In some regions of RB1 a single amino
acid loss:
rise to mild retinoblastoma or
incomplete penetrance
 BMD patients:
Some times in-frame
deletions/duplications
DMD deletions:
 mostly disrupt the reading frame
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Deletions/insertions/duplications
In untranslated regions:
these might affect
transcription/expression and/or stability of
the message:
Fragile X
MD expansions.
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Mutation
Databases
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Mutation Databases
Online Mendelian Inheritance in Man
(OMIM)
problem of collecting mutations
if each out of approximately 50 000
genes can be subject to 100 mutations to
cause disease
then there could be potentially five million
mutations
it needed to get organised quickly to
undertake
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Examples of central and locusspecific databases
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Current mutation
detection
methods
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Characteristics
of the scanning
methods
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