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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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