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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