Highthrough-out put microsatellite allele sizing with high resolution

http://crop.scijournals.org/cgi/reprint/43/5/1828
Published in Crop Sci. 43:1828-1832 (2003).
© 2003 Crop Science Society of America
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GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY
A Low-Cost, High-Throughput Polyacrylamide Gel Electrophoresis System for Genotyping with Microsatellite DNA Markers

D. Wang, J. Shi, S. R. Carlson, P. B. Cregan, R. W. Ward, and B. W. Diers*
Microsatellite DNA markers are widely used in genetic research. Their use, however, can be costly and throughput is sometimes limited. The objective of this paper is to introduce a simple, low-cost, high-throughput system that detects amplification products from microsatellite markers by nondenaturing polyacrylamide gel electrophoresis. This system is capable of separating DNA fragments that differ by as little as two base pairs. The electrophoresis unit holds two vertical 100-sample gels allowing standards and samples from a 96-well plate to be analyzed on a single gel. DNA samples are stained during electrophoresis by ethidium bromide in the running buffer. In addition, one of the gel plates is UV-transparent so that gels can be photographed immediately after electrophoresis without disassembling the gel-plate sandwich. Electrophoresis runs are generally less than two hours. The cost per gel, excluding PCR cost, is currently estimated at about $2.60, or less than $0.03 per data point. This system has been used successfully with soybean [Glycine max (L.) Merr.] and wheat (Triticum aestivum L.) microsatellite markers and could be a valuable tool for researchers employing markers in other species.


Abbreviations: bp, base pair • ITMI, International Triticeae Initiative • PCR, polymerase chain reaction • QTL, quantitative trait loci • SSR, simple sequence repeat



http://www.plantmethods.com/content/1/1/3
High throughput, high resolution selection of polymorphic microsatellite loci for multiplex analysis
Nicholas C Cryer1 , David R Butler2 and Mike J Wilkinson1

1School of Biological Sciences, University of Reading, Reading, Berkshire, RG6 6AS, UK

2Cocoa Research Unit, The University of West Indies, St. Augustine, Trinidad and Tobago

author email corresponding author email

Plant Methods 2005, 1:3doi:10.1186/1746-4811-1-3

The electronic version of this article is the complete one and can be found online at: http://www.plantmethods.com/content/1/1/3

Received: 25 May 2005
Accepted: 18 August 2005
Published: 18 August 2005

© 2005 Cryer et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Keywords: Multiplex, Microsatellite, High Throughput, Fluorescent, Dinucleotide, High-Resolution, Allelic Ladder

Abstract
Background
Large-scale genetic profiling, mapping and genetic association studies require access to a series of well-characterised and polymorphic microsatellite markers with distinct and broad allele ranges. Selection of complementary microsatellite markers with non-overlapping allele ranges has historically proved to be a bottleneck in the development of multiplex microsatellite assays. The characterisation process for each microsatellite locus can be laborious and costly given the need for numerous, locus-specific fluorescent primers.

Results
Here, we describe a simple and inexpensive approach to select useful microsatellite markers. The system is based on the pooling of multiple unlabelled PCR amplicons and their subsequent ligation into a standard cloning vector. A second round of amplification utilising generic labelled primers targeting the vector and unlabelled locus-specific primers targeting the microsatellite flanking region yield allelic profiles that are representative of all individuals contained within the pool. Suitability of various DNA pool sizes was then tested for this purpose. DNA template pools containing between 8 and 96 individuals were assessed for the determination of allele ranges of individual microsatellite markers across a broad population. This helped resolve the balance between using pools that are large enough to allow the detection of many alleles against the risk of including too many individuals in a pool such that rare alleles are over-diluted and so do not appear in the pooled microsatellite profile. Pools of DNA from 12 individuals allowed the reliable detection of all alleles present in the pool.

Conclusion
The use of generic vector-specific fluorescent primers and unlabelled locus-specific primers provides a high resolution, rapid and inexpensive approach for the selection of highly polymorphic microsatellite loci that possess non-overlapping allele ranges for use in large-scale multiplex assays.


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http://www.biotechniques.com/biotechniques/BiotechniquesJournal/2007/April/Microsatellite-marker-identification-using-genome-screening-and-restriction-ligation/biotechniques-41700.html?autnID=588199

Microsatellite marker identification using genome screening and restriction-ligation

Helena Korpelainen, Kirsi Kostamo, Viivi VirtanenUniversity of Helsinki, Helsinki, FinlandBioTechniques, Vol. 42, No. 4, April 2007, pp. 479–486 Full Text (PDF) Supplementary MaterialKorpSUPP424 (.pdf)


http://www.biotechniques.com/biotechniques/multimedia/archive/00010/97225pf01_10994a.pdf
Agarose-Based System for Separation of Short Tandem Repeat Loci


http://www.academicjournals.org/AJB/PDF/pdf2009/3Jun/Wang%20et%20al.pdf
A new electrophoresis technique to separate microsatellite alleles - QiAxcell System