1 / 14

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

libby-reynolds
Télécharger la présentation

Accessing Genetic Variation: Genotyping Single Nucleotide Polymorphisms

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related