PCR-generated artefact from 16S rRNA gene-specific primers

Area of Science:

• Microbiology
• Molecular Biology
• Bioinformatics

Background:

• Polymerase chain reaction (PCR) is crucial for amplifying bacterial 16S rRNA genes.
• Terminal Restriction Fragment Length Polymorphism (TFLP) analysis relies on accurate PCR amplification.
• Commonly used primer pairs can sometimes produce artefactual sequences.

Purpose of the Study:

• To identify and characterize artefacts generated during 16S rRNA gene PCR.
• To investigate the origin of these artefacts in microbial community analysis.
• To provide a solution for avoiding artefactual sequence formation.

Main Methods:

• Performing PCR amplification of bacterial 16S rRNA genes with a standard primer pair.
• Analyzing PCR products using TFLP for microbial community analysis.
• Sequencing amplified products to identify artefactual motifs.
• Searching GenBank for similar repetitive sequences.

Main Results:

• Concatenated oligonucleotide primer sequences were generated as artefacts during suboptimal PCR.
• These artefacts were observed in both complex microbial communities and pure cultures.
• Similar repetitive motifs were identified in existing GenBank sequences.
• The artefacts were linked to the specific primer pair used.

Conclusions:

• Suboptimal PCR conditions with specific primers can lead to artefactual sequence generation.
• These artefacts can impact the accuracy of T-RFLP and other molecular analyses.
• Alternative primer selection is recommended to avoid artefact formation.
• Careful primer choice is essential for reliable microbial community profiling.