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Microsoft PowerPoint ABRF tutorial 2003 update

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FARG Tutorial 2003. Part 2. Single Base Extension: A Method .... Sap and Exo l Treatment. 45 min incubation time. 1 step process ...

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Microsoft PowerPoint ABRF tutorial 2003 updatePage 1FARG Tutorial 2003Part 2Single Base Extension: A Method for Detecting Multiple SNPs Using Automated DNA SequencersRebecca Scholl University of UtahPage 2Objective Discuss the the basics of Single Base Primer Extension Primer selection Examples using the ABI PrismSNaPshot ddNTP Primer Extension Kit Chemistry and cleanup methods Sample electrophoresis: Gels v. CapsPage 3Single Base Extension: Advantages Base discrimination at the target SNP Homozygous and Hetrozygous status Can be cheaper than conventional sequencing for multiple SNP sites Sense and antisense strands can be evaluated simultaneously for confirmation of SNP Can be run on any conventional DNA sequencer available in most labs. No new equipment costs Sequencing dyes used Allows verification of SNP before high throughput screeningPage 4SNaPshotOverview3 Step Process Amplify PCR fragment containing the SNP of interest Unlabeled primers Perform primer extension sense or antisense primers Fluorescent ddNTP incorporated Visualization of SNPPage 5Template Selection: Amplification of a PCR fragment containing the SNP site Create any sized PCR fragment 30 >500 base pairs Same primers can be used for both amplification steps as long as the primers are specific to the region of interest Multiple SNPs can be contained in one large PCR template for primer extension Plasmid Templates Do not require cleanup before primer extensionPage 6Primer Selection: SNaPshotPrimers Primer is designed to terminate directly 5 of SNP site Primers can be designed in both the forward and reverse directions if conformation is needed Some heterozygous peaks may be easier to see in a different direction depending on the bases Check for normal pitfalls of self priming etc. Primers can be tailed 5 to allow for size multiplexing of the SNPs Allow at least 4 base pairs between products HPLC purify any oligos larger than 30 base pairs to eliminate N1 populations Largest fragment we hav ...

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