SNP Genotyping Santiago C. González-Martínez Center of Forest Research, INIA, PO Box 8111 28080 Madrid, Spain

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Presentation transcript:

SNP Genotyping Santiago C. González-Martínez Center of Forest Research, INIA, PO Box Madrid, Spain

Syvänen (2001) Nature Genetics

DGGE and SSCP

(eco)Tilling

Single-base extension

Fluorescence Polarization with Terminator-Dye Incorporation (FP-TDI) - a method for SNP detection

Chen et al Genome Res. Fluorescence polarization

FP-TDI Assay

What the data might look like… if you are lucky Gene 4CL

Usually, the data is not quite as ‘perfect’… Gene EREBPS

FP-TDI ADVANTAGES No special primers Relatively simple procedure Results are immediate DISADVANTAGES Expensive plate reader = $30, 000 kit = $10, 000 for 100, 000x plates = $6 each cost per reaction = 75c each thermocycler maintenance PCR (ie mostly Taq) costs One SNP per reaction Need to know sequence

Pyrosequencing

Microarrays Syvänen (2001) Nature Genetics

High-throughput methods Syvänen 2005 Nature Genetics

Illumina- BeadStation500G-BeadLab Platform

How does it work?

Experimental Procedure

Example of a “nice” SNP

Examples of midrange SNPs Above threshold: GT = Cluster_sep =0.272 Below threshold: GT = Cluster_sep =0.267

4CL-s5[c/t]

AT-s3[g/a] Not resolved with FPBetter separation in Illumina

Illumina- BeadStation demo

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