High throughput, high resolution selection of polymorphic microsatellite loci for multiplex analysis
© Cryer et al; licensee BioMed Central Ltd. 2005
Received: 25 May 2005
Accepted: 18 August 2005
Published: 18 August 2005
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.
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.
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.
KeywordsMultiplex Microsatellite High Throughput Fluorescent Dinucleotide High-Resolution Allelic Ladder
Microsatellite analysis using fluorescently labelled primers and capillary fractionation is the pre-eminent method for the genetic analysis of eukaryotic organisms. The approach is routinely used for many applications including forensic analysis , linkage mapping and association genetics , population genetics [3, 4] and genetic analysis of diversity . The need to screen microsatellite loci for polymorphism between genotypes within the target organism, and for their suitability in multiplex analysis, is an inevitable part of such efforts. The high cost of fluorescently labelled primers has meant that selection of microsatellite markers has typically relied on initial, low-resolution screens of unlabelled primers prior to high-resolution marker selection using labelled primers. However, the preliminary screen is inevitably crude and inefficient, making it either prone to error or reliant upon the more expensive high-resolution selection. There is therefore a need for a high throughput and high-resolution single-step method of selecting appropriate microsatellite markers for genetic studies .
For multiplex analysis, greatest efficiency is achieved when utilising many polymorphic loci possessing closely spaced, non-overlapping allelic ranges. Unexpected allelic range overlap between multiplexed microsatellite loci yields ambiguous alleles that may be misassigned to an inappropriate locus, compromising the integrity of the data set. One inevitable problem lies in the possibility that the screen does not encompass all alleles present in the population under study. Confidence in the definition of allelic ranges is invariably a function of the number and diversity of genotypes screened. There is therefore a balance between the desire to examine many individuals and the cost of doing so using fluorescently labelled primers. Thus, screening invariably becomes expensive as the number of genotypes tested grows, and as the number of discarded markers increases. Common approaches to selecting microsatellite markers for multiplex use include assembling panels from previously fluorescently characterised individual markers [7, 8], and pre-screening markers on polyacrylamide gels utilising radioactivite labelling of PCR products . Several authors have proposed low cost alternatives for preliminary screens using direct DNA staining following polyacrylamide gel electrophoresis [10–13]. Such strategies have merit, but are labour-intensive, cannot assign actual size ranges and generally lack the resolution required to accurately predict polymorphism in dinucleotide markers [10–13]. One methodology that is able to generate high resolution allelic ladders in a similar fashion to the method reported here is that of Oetting . This method employs the use of locus specific primers tailed with generic sequence allowing a second round of labelled PCR and subsequent capillary fractionation. This method however suffers from a number of potential disadvantages relative to our method. The use of long oligonucleotides for PCR of genomic templates at below optimal annealing temperature allows for an increased frequency in the production non specific amplification products. The PCR amplification conditions required for locus specific amplification using tailed oligonucleotides are often different to those conditions optimal for amplification with equal length 20 mer oligonucleotides. The method of Oetting is not suited to the genotypic analysis of dinucleotide repeat markers due the possibility of extensive stutter profiles generated by the second round of PCR complicating the allelic profiles and so is only considered of merit for marker selection.
Here, we propose a simple but novel approach in which microsatellite amplicons generated from pooled genomic DNA templates are ligated into a standard cloning vector, re-amplified using a labelled universal primer targeting the plasmid insert flanking region, and an unlabelled locus-specific primer. The resultant profiles represents allelic ladders derived from the component alleles contained by the pooled DNA. This procedure thereby offers a single assay, high resolution and inexpensive means of screening microsatellite loci for polymorphism and allelic size range. The profile also offers a qualitative indication of the locus with regard to stutter, a problem often associated with the use of dinucleotide repeat markers for genetic analysis, but also of interest to laboratories utilising tri- and tetranucletide repeat markers.
Comparison of predicted and observed microsatellite allele frequency. The number of alleles each locus was predicted to generate as described in Figure 1 was compared to the actual alleles observed when screened over 672 genotypes of wild, uncultivated cocoa. Predictions were based on the height and number of peaks reported from the pooled samples and took account of the extra DNA amplified from the pDrive vector when predicting the size of the DNA fragments.
Adoption of this methodology allows for both a qualitative and semi quantitative characterisation of polymorphism at individual microsatellite loci. When using pooled samples, combining DNA from up to 12 individuals allowed for the reliable detection of single copy alleles within that sample. If characterising microsatellite loci using DNA pools of greater than 12 individuals the incorporation of a buffer zone into the final genotyping assay, based on the observed range of allele sizes, can allow for the variability likely to be encountered in a larger sample size. The methodology is suitable for high throughput applications by the combination of differing fluorescent dyes in association with convenient liquid handling formats. This protocol benefits from initially utilising unlabelled primers identical to those used in the final genotyping assay, reducing the possibilities of unexpected banding patterns due to changes in primer sequence or assay conditions. The high resolution DNA size measurement makes this protocol suitable for characterising dinucleotide microsatellite loci.
List of abbreviations
Polymerase chain reaction
We thank Olivier Sounigo, and Claire Lanaud for providing DNA, Didier Clement for providing as then unpublished microsatellite information (Pugh et al., 2004) and Steve Brown for providing a linkage map of cocoa microsatellite markers. This work forms part of a collaboration between The University of Reading and the Cocoa Research Unit, The University of West Indies, Trinidad and Tobago, funded by The Biscuit, Cake, Chocolate and Confectionery Association, United Kingdom.
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