METHODS FOR EXTENDED MOLECULAR HALOTYPING

Yale University, New Haven CT

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Abstract

Linkage disequilibrium analysis is an essential tool in refining the locations of disease genes and isolating such genes by positional cloning, and is likely to increase in importance as the sequence of the human genome becomes more complete. Understanding what normal variation in people and what drives variation is critical for a complete understanding of the information that the sequence provides. Yet very little is known about linkage disequilibrium in different populations or even in different individuals over large genomic regions. The overall objective of this project is to develop novel methods to examine extended molecular haplotypes and to obtain accurate physical mapping distances for relevant polymorphic markers for analysis of recombination and genetic disequilibrium over large genomic distances. Several essential elements of positional cloning-molecular haplotyping to set phase, discovery of new polymorphic markers, and analysis of physical distances between markers in disease gene regions-are time consuming and laborious. Our initial goal is to develop new methods for haplotype analysis and regional mapping that are based on the use of isothermal amplification and single-stranded cloning vectors. Their utility will be examined using a 1 Mb or more regions on the p arm of chromosome 6 that roughly centers on the hemochromatosis gene for which there is a well-defined clone contig with numerous polymorphic markers. The region contains a domain at the extreme centromeric end that is at least partially refractory to cloning due, apparently, to the presence of alpha satellite repeat sequences with an adjacent segment of DNA that is represented at a number of other sites in the genome. This will present a 'real' test of the application of linkage disequilibrium methods and theory. Clusters of 3-4 polymorphic markers, encompassing 20-30 kb, will be examined in African (Biaka), mixed European, and AmerIndian (PIMA) populations, with the cluster spaced at intervals of 80-100 kb over 1Mb. This will allow estimates of disequilibrium at intermediate distances (70-100 kb), where recombination effects may be significant, as well as at short distances (8-10 kb). The choice of marker spacing is optimal both for analysis of extended molecular haplotypes and for the development of methods of trapping genomic DNA strands from test individuals in an allele-specific manner, since it provides two target sizes (10 kb and 100 kb). Retrieval of allele-specific DNA segments from individuals by allele-specific amplification and affinity-purification may provide substrates for more rapid analysis of genomic regions for both basic research and positional cloning.