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Open Access Methodology article

g you The direct determination of haplotypes from extended regions of genomic DNA

David Stirling1 and Michael J Stear2*

Author affiliations

1 Department of Haematology, Royal Infirmary of Edinburgh, Little France Crescent, Edinburgh EH16 4SA, UK

2 Division of Animal Production and Public Health, Faculty of Veterinary Medicine, Glasgow University, Bearsden Road, Glasgow G61 1QH, UK

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Citation and License

BMC Genomics 2010, 11:223  doi:10.1186/1471-2164-11-223

Published: 6 April 2010

Abstract

Background

One of the major obstacles to the exploitation of genetic variation in human medicine, veterinary medicine, and animal breeding is the difficulty in defining haplotypes in unrelated individuals.

Results

We have developed a Multiplex Double Amplification Refractory Mutation System combined with Solid Phase PCR on Fluorescently labelled beads. The process is inherently amenable to automation. It provides a high degree of internal Quality Control, as each PCR product is represented in duplicate on the bead array, and each SNP is tested against multiple partners. This technique can resolve very complex genotypes into their constituent haplotypes; it defined all the alleles at 60 SNP in exon 2 of the ovine DRB1 MHC locus in a sample of 109 rams. These 60 SNP formed 33 DRB1 exon 2 alleles; two of which had not been previously identified; although both of them have been independently confirmed.

Conclusion

This technique has the same resolution as allele specific sequencing. Sequencing has the advantage of identifying novel polymorphic sites but where all SNP sites have been identified this novel procedure can resolve all alleles and haplotypes and identify novel combinations of polymorphisms. This method is similar in price to direct sequencing and provides a low cost system for direct haplotyping of extended DNA sequences.