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1 Methods for detection of un known mutations BRCA 2 BRCA1 Gene 3 BRCA2 Gene 4 SSCP single strand conformation polymorphism simplicity clearly by heteroduplex analysis (HA) 5 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 6 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 7 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. 8 Pedigree of a selected family with breast cancer 9 SSCP Analysis BRCA1 Exon 15, 4650delCA 10 Pedigree of a selected family with breast cancer 11 SSCP Analysis BRCA1, Exon 20, Nt 5382 12 SSCP Analysis Exon 11pi BRCA1 MS R1347G 13 Protein truncation test PTT 14 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 15 PTT Coding sequence without introns cDNA via RT-PCR from RNA or large exons in genomic DNA 16 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 17 Protein truncation test (PTT) 18 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 19 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. 20 Protein truncation test used in diagnostic laboratories dealing with cancer genes because they often contain truncating mutations. 21 Protein truncation test (PTT) 22 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 23 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 24 The protein truncation test (PTT) 25 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 26 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. 27 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 28 29 Deletions/insertions/duplications •Out of frame •In frame 30 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 31 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 32 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 33 Deletions/insertions/duplications In untranslated regions: these might affect transcription/expression and/or stability of the message: Fragile X MD expansions. 34 Mutation Databases 35 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 36 Examples of central and locusspecific databases 37 Current mutation detection methods 38 Characteristics of the scanning methods 40 41