1. 2 methods for detection of un known mutations brca
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Methods for Methods for detection of un detection of un known mutationsknown mutations
BRCABRCA
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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 GelsPrepare 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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SSCPLoading Buffer95% 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 conditionsFragment Size: 150-200 bp
6 Watts 10-12 hours room temperature
Fragment Size: > 200 bp 8 Watts 10-12 hours room temperatureExposureDry 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 AnalysisBRCA1 Exon 15, 4650delCA
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Pedigree of a selected family with breast cancer
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SSCP AnalysisBRCA1, Exon 20,Nt 5382
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SSCP AnalysisExon 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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cDNAIt is PCR amplifiedThe forward primer carries at its 5'
end a T7 promoterfollowed by a eukaryotic translation
initiation sequence which includes an ATG start codonNext 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-biotin-alkaline 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 lengthno truncating mutation is presentTruncating mutations result in
shorter productsthe 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 mutationsDeletionsDuplicationsaffecting splicing
Finally, in vitro transcription/translation is used to generate peptide fragmentsanalyzed on SDS-PAGE gelto detect translation terminating
mutations
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The protein truncation test (PTT)
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ADVANTAGES
Detects truncating mutationsAllows 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 genesE.g. APC, BRCA1, BRCA2 and
Dystrophin all have approximately 90-95% 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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DISADVANTAGESThe 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 XMD expansions.
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Mutation Databases
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Mutation Databases
Online Mendelian Inheritance in Man (OMIM)
problem of collecting mutationsif each out of approximately 50 000
genes can be subject to 100 mutations to cause diseasethen there could be potentially five
million mutationsit needed to get organised quickly to
undertake
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Examples of central and locus-specific databases
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Current Current mutation mutation detection detection methodsmethods
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CharacteristicCharacteristics of the s of the
scanning scanning methodsmethods
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