Sequencing of (dA:dT) Cloned Mixed PCR Products from Microbial Populations

Because only between 1 and 10% of bacteria present in soil and aquatic environments are culturable using currently available methods, attempts to identify and quantify the nonculturable, majority population must circumvent the need for culture. Molecular biological techniques, particularly using 16S rRNA sequences, have substantial advantages over traditional culture-based methods for the characterization of natural microbial populations. One particularly interesting strategy involves the use of the polymerase chain reaction (PCR) to amplify target genes from DNA extracted directly from environmental samples. PCR-amplified target sequences can then be cloned to obtain a representative clone bank that reflects the diversity of a target sequence in the original population. After sequencing individual clones, comparative data analysis can be used to assess diversity among a natural population of microorganisms without the need to culture the organisms first.