Genotyping module.

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Presentation transcript:

Genotyping module

Genetic analyzer 3730xl

Capillary electrophoresis

Capillary electrophoresis

Capillary electrophoresis

Capillary electrophoresis

Capillary electrophoresis

Example of genotyping PCR Sample 1: Homozygous for SSR; alleles are the same size, one band on a gel e.g. 120/120 Sample 2: Heterozygous for SSR; alleles are not the same size, 2 bands on gel e.g. 120/126 Biological significance??

Gene Mapper Software GeneMapper is a flexible genotyping software package that provides DNA sizing and quality allele calls for all Applied Biosystems electrophoresis-based genotyping systems. Applications: amplified fragment length polymorphism (AFLP), loss of heterozygosity (LOH), microsatellite, and SNP genotyping.

Capillary electrophoresis

Creating a customized project KIT Cassava SSR PANEL Co-load group 1 MARKERS M1 (PET)/ M2 (VIC)/ M3 (6-FAM)/ M4 (NED)

Failed PCR

Non-specific PCR

Primer dimers

Pull ups

A+/A- hybrid

Stuttering

1 marker + Liz 500

1 marker

1 marker- 4 alleles

Configuring panels Defn Panel: A set of markers that can be loaded together Considerations: Load markers of different size lengths Distinguish different markers from those with overlapping size ranges by using dye labels Use more PCR prdt labeled with dyes of low emission intensity than dyes of high emission intensity. 6-FAM (blue), VIC (green) > NED (black) > PET (red) > LIZ (orange)(highest to lowest) Liz 500 is the size standard; range: 35 - 500 bp, 16 single- strand labeled fragments (35, 50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490, & 500 bases).

Co loading strategy

Co loading strategy

Co loading strategy