Microbial community analysis — In PCR-based cloning/sequencing of mixed rRNA gene amplicons, why is a cloning step included before sequencing?

Introduction / Context:
Environmental microbiology often amplifies rRNA genes from complex communities to profile diversity. Before high-throughput sequencing was widespread, cloning PCR products into plasmids and sequencing individual clones was the standard approach. Understanding the rationale for the cloning step clarifies how heterogeneous mixtures are resolved into individual sequences.

Given Data / Assumptions:

• We have a mixed PCR product containing rRNA gene fragments from many organisms.
• Sequencing directly would produce overlapping chromatograms (in Sanger methods) that cannot be interpreted.
• Cloning into vectors allows isolation of single inserts for clean reads.

Concept / Approach:
Cloning distributes heterogeneous amplicons among individual plasmids, each carried by a separate colony. Picking single colonies ensures each sequencing reaction reads one unique insert, thereby separating the mixture into resolvable components. This step is not primarily to “improve amplification” or to “prevent contamination,” although good technique reduces contamination. Modern amplicon sequencing (e.g., Illumina) uses barcodes rather than cloning, but the underlying goal remains the same: deconvolute sequence mixtures.

Step-by-Step Solution:

Verification / Alternative check:
Sanger chromatograms from mixed templates show overlapping peaks; after cloning, chromatograms are clear and single-peaked, confirming separation.

Why Other Options Are Wrong:

• The amplification process: cloning does not amplify; PCR already does.
• Preventing contamination: sterile technique and controls address contamination, not cloning per se.
• None of these: incorrect because separation of mixed sequences is the point.

Common Pitfalls:
Assuming cloning is required for PCR success. It is required for resolving mixtures into single sequences when using Sanger sequencing of complex samples.

Final Answer:
separating the different rRNA gene sequences in the mixture