Heteroduplex mobility assay as a tool for predicting phylogenetic affiliation of environmental ribosomal RNA clones.
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.
Bowyer
J
J
Department of Biological Sciences, Commonwealth Key Centre for Biodiversity and Bioresources, Division of Environmental and Life Sciences, Macquarie University, NSW 2109, Sydney, Australia.
Verrills
N
N
Gillings
M R
MR
Holmes
A J
AJ
D001419:Bacteria / Q000145:classification* / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis; D004783:Environmental Microbiology; D020180:Heteroduplex Analysis; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S