Journal: Applied and Environmental Microbiology
Article Title: Formation of Pseudo-Terminal Restriction Fragments, a PCR-Related Bias Affecting Terminal Restriction Fragment Length Polymorphism Analysis of Microbial Community Structure
Figure Lengend Snippet: (A to C) T-RFLP analysis of clone PeH59 (affiliated with the CFB phylum) amplicons after restriction digestion with different enzymes, resulting in the expected T-RFs only ( Msp I [A]) or in the formation of pseudo-T-RFs ( Alu I [B] and Hha I [C]). (D) 16S rRNA gene secondary structure of clone PeH59 as predicted by the mfold software including the sequence stretches around detected pseudo-T-RFs. RS, restriction sites. Bold numbers indicate restriction sites with corresponding T-RFs in the electropherogram. RFU, relative fluorescence units.
Article Snippet: DNA (75 ng for amplicons from the gut DNA extract, 50 ng for clonal amplicons), 2.5 U of restriction enzymes ( Msp I, Taq I, and Alu I [Promega, Mannheim, Germany]; Msp I, Hpa II, Hha I, Hae III, and Bst UI [New England Biolabs, Frankfurt am Main, Germany]; and BsiS I [Minotech Biotechnology, Heraklion, Crete, Greece]), 1 μl of 10× incubation buffer, and 1 μg of bovine serum albumin (if recommended) were combined in a total volume of 10 μl and digested for 3 h at 37°C ( Msp I, Alu I, Hpa II, Hha I, and Hae III), 55°C ( Bsi SI), 60°C ( Bst UI), 65°C ( Taq I), or 70°C ( Bsi SI).
Techniques: Software, Sequencing, Fluorescence