High-Throughput Production of Optimized Primers (Fimers) for Whole-Genome Direct Sequencing

Fimers are specifically modified primers selected to inhibit nonspecific interactions occurring in cycle sequencing. They are postsynthetically derived from 2′-methoxyoxalamido or 2′-succinimido precursor oligonucleotides by treatment with appropriate small molecular weight modifiers (a primary aliphatic amine or hydroxide anion). We describe design, synthesis, and purification of fimers, and their use in protocols for direct sequencing of genomic deoxyribonucleic acid (DNA). Protocol for isolation of microbial genomic DNA is also reported.