High throughput, high resolution selection of polymorphic microsatellite loci for multiplex analysis

Plant Methods

Mike J Wilkinson¹  David R Butler²  Nicholas C Cryer¹ 

[1] School of Biological Sciences, University of Reading, Reading, Berkshire, RG6 6AS, UK;Cocoa Research Unit, The University of West Indies, St. Augustine, Trinidad and Tobago

关键词: Allelic Ladder;    High-Resolution;    Dinucleotide;    Fluorescent;    High Throughput;    Microsatellite;    Multiplex;   

Others  :  1218692
DOI  :  10.1186/1746-4811-1-3

 received in 2005-05-25, accepted in 2005-08-18,  发布年份 2005

【 摘 要 】

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.

【 授权许可】

   
2005 Cryer et al; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

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