Formation of pseudo-terminal restriction fragments, a PCR-related bias affecting terminal restriction fragment length polymorphism analysis of microbial community structure

Abstract

Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called "pseudo-T-RFs" since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.

FIG. 1.

Occurrence of pseudo-T-RFs in T-RFLP profiles of a single clone depending on the restriction enzyme used. 16S rRNA gene T-RFLP electropherograms were derived from clone PeM75 (affiliated with Lactobacillales). Numbers indicate restriction sites (RS) for the respective enzyme detected in the clonal sequence between bases 1 and ∼900 (length of the PCR product), counted from the labeled 5′ end. Bold numbers indicate restriction sites with corresponding T-RFs in the electropherogram. RFU, relative fluorescence units.

FIG. 2.

Effect of mung bean nuclease digestion on the occurrence of pseudo-T-RFs in T-RFLP profiles (AluI digests) of environmental, clonal, and pure-culture samples. Insets show the T-RFLP profile after mung bean nuclease digestion. The number of PCR cycles used to produce the amplicons is indicated. Fragment lengths of pseudo-T-RFs are shown in bold. Clone PeMAr04 is affiliated with the kingdom Crenarchaeota. MB, Methanobactericeae; CR, Crenarchaeota; RFU, relative fluorescence units.

FIG. 3.

(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 (MspI [A]) or in the formation of pseudo-T-RFs (AluI [B] and HhaI [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.

FIG. 4.

Effect of restriction digest temperature on the formation of pseudo-T-RFs of clone PeH59. Restriction digests were performed using BsiSI at 55°C (A) and 70°C (B) and by using a 3-min denaturation of the PCR amplicon prior to the addition of enzyme and incubation at 70°C (C). Bold numbers indicate restriction sites with corresponding T-RFs in the electropherogram. RFU, relative fluorescence units.

FIG. 5.

Effect of the position of the terminal restriction site on the extent of pseudo-T-RF formation, based on in vitro T-RF formation of 56 bacterial clones with MspI as the restriction endonuclease. The peak area of the pseudo-T-RF is compared to the peak area of the primary T-RF and given as a percentage. Clones were obtained from a 16S rRNA gene clone library derived from the midgut of cetoniid beetle larvae (Egert et al., unpublished).

FIG. 6.

Effect of PCR cycle number on the extent of pseudo-T-RF formation observed with amplicons of clone PeM75 after MspI digestion. The peak area of the pseudo-T-RF is compared to the peak area of the primary T-RF and given as a percentage. Error bars (which represent standard deviation) are based on three replicates.

FIG. 7.

Schematic model of pseudo-T-RF formation. (A) PCR-related parameters influencing the formation of partly single-stranded amplicons. (B) Involvement of the secondary structure of partly single-stranded amplicons in the formation of pseudo-T-RFs. dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; solid triangles, restriction site cut (MspI); open triangle, restriction site not cut.