By Pui-Yan Kwok
Pui-Yan Kwok, MD, PhD, has assembled a complete selection of without problems reproducible concepts for the tricky means of unmarried nucleotide polymorphisms (SNP) discovery and genotyping. those state-of-the-art protocols for mutation/SNP detection make the most of denaturing high-performance liquid chromatography (dHPLC), single-strand conformation polymorphism (SSCP), conformation-sensitive gel electrophoresis (CSGE), chemical cleavage, and direct sequencing. both robust and up to date equipment are given for genotyping SNPs, together with molecular beacons, the Taqman assay, single-base extension techniques, pyrosequencing, ligation, the Invader assay, and primer extension with mass spectrometry detection.
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Additional info for Single Nucleotide Polymorphisms: Methods and Protocols
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3. 8: 90 mM Tris base, 90 mM boric acid, 2 mM Na2EDTA, 10% glycerol. 4. Size marker: GeneScan 500 TAMRA (Applied Biosystems), purified by ultrafiltration using Microcon 50 (Millipore, Bedford, MA) to remove ions and low molecular-weight fragments, and adjusted to half its original volume. 5. Deionized formamide (stored at –20°C in aliquots). 6. Separation matrix: GeneScan Polymer (Applied Biosystems) in 1X TBEG. GeneScan Polymer is supplied as a 7% solution, which is diluted to 6% by mixing with 20X TBE and glycerol (see Note 2).
Am. J. Hum. Genet. ), A266. 2. Xiao, W. and Oefner, P. J. (2001) Denaturing high performance liquid chromatography: a review. Human Mutat. 17, 439–474. 3. Jones, A. , Oefner, P. , Cheadle, J. , and O’Donovan, M. C. (1999) Optimal temperature selection for mutation detection by denaturing HPLC and comparison to single-stranded conformation polymorphism and heteroduplex analysis. Clin. Chem. 45, 1133–1140. 4. Spiegelman, J. I. , Mindrinos, M. , and Oefner, P. J. (2000) Highaccuracy DNA sequence variation screening by dHPLC.
Cut the relevant section of the gel with a scalpel and lift the gel section with a piece of dry Whatman 3MM blotting paper. 3. Release the gel section on the transilluminator by wetting the filter paper with water. 4. Photodocument the ethidium bromide stained bands under transillumination with an orange-red color correction filter using Polaroid type 667 film. 7. CSGE Using Radioactive PCR Products The method of CSGE can also be adapted to using radioactive PCR products that remove the need for photography for gel documentation.