DeCode Genetics Inc. is Icelandic company studying the genome of about 250,000 Iclendic people.  The company's approach is to genotype microsatellite markers (STS) and find genetic linkage between disease phenotype and region in human genome.  The region identified by linkage analysis usually contains several genes and may be up to 10,000,000 bases long.  In order to find disease causative mutation it is necessary to compare genes sequences between affected (with disease) and non-affected (control) individuals in the linked region.  I propose a method to amplify large genomic regions as a set of overlapping long and accurate PCR (LA-PCR) fragments (see figure below).  The project had several technichal challenges.  I have optimized LA-PCR buffer conditions to improve yields of the reaction.  The buffer was essentially as described in Barnes W.M. 1994 with addition of 0.1 mg/ml acetylated BSA (NEB Cat. # B9001S).  Eva Halapi has found the method to clone large PCR fragments by using TOPO TA cloning kit from Invitrogen (Cat. No K452001).  Upon sequencing of cloned large PCR fragments we found that LA-PCR amplification from diploid human genome may yield chimeric molecules contatining multiple recombinations between to chromosome copies.  To overcome this pitfall we used allele-specific primers compentary to single nucleotide polymorphism in the genomic region of interest.  Such primers amplified the LA-PCR fragment from only one chromosome.  Later at InforMax Inc., I have developed the computer algorithm to design primers for this method.









Figure depicting primer design algorithm for amplification of large genomic regions as set of overlapping LA-PCR fragments