Published online 2 August 2005
Methods Online |
Simultaneous detection of microsatellite repeats and SNPs in the macrophage migration inhibitory factor (MIF) gene by thin-film biosensor chips and application to rural field studies
1Department of Genetics, Yale University School of Medicine 333 Cedar Street, New Haven, CT 06510, USA 2Department of Medicine, Yale University School of Medicine 333 Cedar Street, New Haven, CT 06510, USA 3Department of Epidemiology and Public Health, Yale University School of Medicine 333 Cedar Street, New Haven, CT 06510, USA 4Department of Molecular, Cellular and Developmental Biology, Yale University School of Medicine 333 Cedar Street, New Haven, CT 06510, USA 5Department of Pathology, Yale University School of Medicine 333 Cedar Street, New Haven, CT 06510, USA 6Thermo Electron, Corp., Clinical Chemistry Division Louisville, CO 80027, USA 7Department of Internal Medicine, Kangnam St Mary's Hospital, The Catholic University of Korea Seoul, Korea 8North Shore-LIJ Research Institute Manhasset, NY, USA 9Macha Malaria Research Institute Choma, Zambia 10Macha Malaria Research Institute Dillsburg, PA, USA
*To whom correspondence should be addressed. Tel: +1 203 737 1453; Fax: +1 203 785 7053; Email: Richard.Bucala{at}Yale.edu
Received June 8, 2005. Revised July 14, 2005. Accepted July 14, 2005.
Microsatellite repeat and single nucleotide polymorphisms (SNPs) are abundant sources of genetic variation, but existing methodologies cannot simultaneously detect these variants in a facile or inexpensive way. We describe herein a thin-film biosensor chip based on an allele-discriminating oligonucleotide array that enables genotyping for both microsatellite repeats and SNPs in a single analysis. We validated this methodology for the functionally polymorphic –794 CATT5–8 repeat and –173 G/C SNP present in the promoter of the human gene for macrophage migration inhibitory factor (MIF). In a comparison of 30 samples collected at a rural hospital in Zambia, we observed a 100% concordance for both the CATT repeat and G/C SNP between the biosensor methodology and the conventional capillary electrophoresis. The biosensor chips are low in cost and once printed, they are robust and require no instrumentation for analysis. When combined with multiple displacement amplification, this methodology can be utilized in primitive settings for the genotyping of nanogram quantities of DNA present in blood, dried and stored on filter paper samples. We applied this methodology to a field study of MIF genotype in children with malaria, and provide first evidence for a potential association between MIF alleles and malaria infection. We also present data supporting significant population stratification of the low- versus high-expression forms of MIF that may bear on the role of this gene in infectious diseases.
Present addresses: Xiao-bo Zhong, Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA
Patricia Bray-Ward and David C. Ward, Nevada Cancer Institute, 10000 W. Charleston Boulevard, Las Vegas, NV 89117, USA
Correspondence may also be addressed to Xiao-bo Zhong. Tel: +1 913 588 0400; Fax: +1 913 588 7501; Email: xzhong{at}kumc.edu
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