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Accessing Genetic Variation: Genotyping Single Nucleotide Polymorphisms

Accessing Genetic Variation: Genotyping Single Nucleotide Polymorphisms. February 24, 2004. Syvänen, Nature Genetics 2001:930-942. What is Genotyping?. Currently, the focus has changed from SNP discovery to genotyping individuals

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Accessing Genetic Variation: Genotyping Single Nucleotide Polymorphisms

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  1. Accessing Genetic Variation: Genotyping Single Nucleotide Polymorphisms February 24, 2004 Syvänen, Nature Genetics 2001:930-942

  2. What is Genotyping? • Currently, the focus has changed from SNP discovery to genotyping individuals • Using genotyping we can correlate linkage disequilibrium (LD)- “Genes not in random association” Hartl and Clark -1997 (pg 96), in order to find a correlation of multifactorial traits to candidate genes. • Genotyping follows two formats - the assay used as well as the detection method

  3. Recent techniques used to map locations using SNP genotypes • This table demonstrates the number of SNPs and samples necessary to do population genetics or associate linkage disequilibrium

  4. Flow chart for Genotyping

  5. Hybridization Methods • Hybridization of probe is extremely stringent at optimal conditions (false positives) • Semi-homogenous (Chemiluminescence or fluorescence) • Solid-phase microarrays (Fluorescence) • Solution-phase homogeneous (Fluorescence polarization or FRET)

  6. How does hybridization work with Real-Time Molecular Beacon probe TaqMan probe Allele Fluorescence profile

  7. Real Time Detection • This figure gives a better overview of how both TaqMan and Molecular Beacons bind at a site and release fluorescence, if the SNP corresponds to the probe • Great for multiplexing TaqMan Molecular Beacon Kirk et al. 2002 (Fig 2)

  8. Primer Extension • Follows every assay format except solid-phase microarrays

  9. Microarray primer extension • Can detect heterozygosity • Potential rate of false positives

  10. Primer Extension • Pyrosequencing - uses pyrophoshates, apyrase, and luciferase to measure the exact base pair sequence incorporated after the sequencing primer • Can get up to 50 base pairs • Can not multiplex, plus very costly www.pyrosequencing.com

  11. Allele-specific ligation probes • Whether ligation occurs, depends on SNP • If probes matched perfectly, then they will be joined with a ligase, in which the SNP can be categorized • Potential multiplexing capabilities • Solid-phase microtiter plate (indirect colorimetric or FRET)

  12. Allele-specific PCR • Uses primary allele primer and secondary probe to create fluorescence for SNP detection • Primers very expensive • Based on the idea of FRET (Fluorescence Resonance Energy Transfer)

  13. Endonuclease Cleavage RFLP Invader probe • Invader probe helps cleavage by Flap endonuclease, which emits fluorescence detected by Mass Spec • Uses solution-phase homogeneous (Fluorescence polarization or FRET)

  14. Conclusion • Each technique has its own unique ability to detect SNPs depending on the assay or detection method • One must weigh factors such as: • Cost • Throughput • Simplicity of assay design • Multiplexing • Sensitivity/accuracy • After all these factors are analyzed then you can pick the correct method for your SNP detection

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