Dna Sequencing

Significant progress, particularly in working with environmental samples, has been made since the dideoxy chain termination method for DNA sequencing was first described by Sanger et at. (1977). The advent of fluorescent dyes, improvements in gel matrix technology, cycle sequencing using a thermal cycler, capillary systems, use of lasers, and automated gel analysis now allows up to 1100 bases of sequence to be generated or deduced in a single reaction. Manual sequencing is extremely time-consuming and is very rarely performed in individual laboratories any more. It is more cost effective to send sample DNA to a commercial sequencing facility for analysis.

Part of the usefulness of DNA sequencing lies in determining gene sequences of unique or important members of a soil community for use in developing more specific primers and gene probes to address specific ecological questions. Gene sequences, once obtained, are submitted to and maintained within various databases such as GenBank (http://www.ncbi.nlm.nih.gov/) or the Ribosomal Database Project II (http://rdp.cme.msu.edu/html/). GenBank and its collaborating databases, the European Molecular Biology Laboratory (EMBL; http://www.ebi.ac.uk/embl/) and the DNA Databank of Japan (DDBJ) reached a milestone recently of containing 100 billion bases (100 gigabases) of sequence information from over 165,000 organisms, including bacteria, fungi, protozoa, nematodes, and other fauna. The Ribosomal Database Project II, Release 9.35 (Cole et at., 2005), contains nearly 198,000 aligned and annotated bacterial small subunit (16S) rRNA gene sequences with updated online analyses. Continued development of databases through DNA sequencing is essential and is a prerequisite to good primer and probe design.

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