CHAPTER 8 COINCIDENCE CLONING: ROBUST TECHNIQUE FOR ISOLATION OF COMMON SEQUENCES

Abstract: Coincidence cloning (CC) is aimed at finding DNA fragments, which are common to the samples under study. The nature of these input DNAs may be genomic or cDNA, cloned or uncloned. The approach is based on cloning identical (or almost identical) nucleotide sequences belonging to different fragmented genomic DNA or cDNA pools, while discarding sequences that are not common to both. Early versions of the CC technique were not very efficient. Their most serious disadvantage was rather low selec-tivity, so that the resulting libraries of the fragments contained large amounts of sequences unique to one of the two sets of DNA fragments under comparison. To avoid this, Azhikina and colleagues were the first to exploit the technique of selective polymerase chain reaction (PCR) suppression, which strongly increased the efficiency of CC. Another important problem is the “nonspecific ” imperfect hybridization between nonorthologous repetitive elements or short sequence-similar sequences, which produces chimeric clones representing an impressive fraction of the libraries (up to 60%, when complex genomic mixtures). An important improvement in this tech-nique comprises treatment of the hybrids with the nucleases, specifically recognizing single-nucleotide mismatches or more extended loop regions. This results in digestion of improperly matched hybrids (primarily chimeras), whereas perfect, nonchimeric het-eroduplexes are greatly enriched in the final mixture (up to 96 % or more)