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Microsatellite Analysis with Elchrom High Resolution Hydrogels
Sponsored,vendor-submitted protocol   Sponsored by Elchrom Scientific AG    Published in BioTechniques Protocol Guide 2008 (p.21)

Introduction

Microsatellites or Short Tandem Repeats (STRs) are DNA sequences with short repetitive units of 1-6 bases, present throughout the whole genome. At the same locus, microsatellites are of different length in different individuals due to different number of repeats. The detection of microsatellites length variability is a very powerful tool used in research, quality control, forensics and diagnostics. By using Elchrom high resolutions Spreadex hydrogels after PCR amplification of microsatellite markers, even small differences in length can be clearly detected. The inert hydrogel matrix allows direct downstream reactions like re-amplification (PCR), sequencing or cloning, without previous purification. Microsatellites applications

Microsatellite analysis can be used for:

  1. Population studies of plants, animals and humans

  2. Paternity tests (Figure 1)

  3. Individual identification in forensics

  4. Monitoring tool during Speed Congenics for animals (Figure 2. GenoMouse)

  5. Quality control in breeding facilities (plant and animal production)

  6. Diagnostics (Figure 3)


Fig 1. (Click to enlarge)



Fig 2. (Click to enlarge)



Fig 3. (Click to enlarge)


Microsatellite amplification by PCR

Simplified PCR protocol allows for high throughput microsatellite analysis: PCR master mix, including DNA, is loaded on a PCR plate. The primers (2x), diluted with Elchrom PCR Blue Dye solution, are added. PCR is started without additional mixing (Web site (external)). Microsatellite analysis with Spreadex hydrogels

High resolving power of Elchrom non-acrylamide hydrogels, allows for highly accurate size based separation and reproducible analysis of microsatellites. After PCR, Microsatellite samples are loaded directly by multichannel pipet and resolved on Elchrom's Spreadex hydrogels. The unit length of microsatellite repeat (di-, tri-, tetra-, etc.) defines the resolution needed to be achieved. The unit length and also the length of the PCR fragments define the optimal type of Spreadex hydrogel and running conditions.

“Find the Best Gel” program will give the optimal Spreadex hydrogel type. “ELQuant” program will show detailed running conditions and optimal gel format ((Web site (external)).

The electrophoresis can be performed either in regular electrophoresis units or in Elchrom specially designed electrophoresis apparatuses: SEA 2000® and ORIGINS by Elchrom™. The electrophoresis gels are stained with standard DNA dyes such as Ethidium Bromide, SYBR Gold, SYBR Green or others. Direct sequencing

The bands of interest are picked out of the hydrogel with Band Pick and mixed with the sequencing reagents. The gel piece containing PCR fragment of interest can be used directly without further purification, due to inert and temperature stable matrix.


Table 1.


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