Heteroduplex mobility assay as a tool for predicting phylogenetic affiliation of environmental ribosomal RNA clones

Jocelyn Bowyer, Nicole Verrills, Michael R. Gillings, Andrew J. Holmes

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Heteroduplex mobility assay (HMA) of partial 16S rRNA gene fragments was tested as a tool for predicting bacterial phylogenetic relationships. Approximately 400-bp fragments were amplified from a selection of cloned environmental DNAs representing a range of sequence identities and phylogenetic relationships. Heteroduplexes between pairs of sequences were formed by mixing equal amounts of PCR products, denaturing and annealing. Annealed mixes were separated on 8% polyacrylamide gels and silver stained. Heteroduplexes were readily distinguished from reannealed homoduplex and unannealed fragments in all sequences where percentage identity was less than 95%. The heteroduplexes showed retarded electrophoretic migration with respect to homoduplexes. The relative retardation was strongly correlated to the percentage sequence identity between the two strands. The HMA is a useful tool for screening environmental clone libraries to systematically select clones representative of the phylogenetic diversity within the sample, or to selectively retrieve members of a particular phylogenetic group for more detailed study.

Original languageEnglish
Pages (from-to)155-160
Number of pages6
JournalJournal of Microbiological Methods
Issue number2
Publication statusPublished - 2000


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