Deconstructing the Polymerase Chain Reaction II: an improved workflow and effects on artifact formation and primer...

Area of Science:

• Microbiology
• Molecular Biology
• Bioinformatics

Background:

• Polymerase chain reaction (PCR) amplification of complex microbial DNA can introduce bias due to inefficient primer-template interactions.
• Previous methods like PEX PCR aimed to reduce this bias by separating PCR stages.
• Degenerate primers, while versatile, can exacerbate bias in complex microbial community analysis.

Purpose of the Study:

• To introduce an improved deconstructed PCR (DePCR) protocol for higher throughput and reduced bias.
• To demonstrate DePCR's effectiveness in decreasing chimeric sequence formation.
• To analyze the impact of annealing temperature on primer utilization and microbial community structure.

Main Methods:

• Implementation of an improved deconstructed PCR (DePCR) protocol separating linear and exponential amplification.
• Utilizing PCR with annealing temperature gradients to assess primer utilization.
• Amplifying microbial DNA with degenerate and non-degenerate primer pools using both DePCR and standard PCR.

Main Results:

• The DePCR protocol significantly reduces chimeric sequence formation compared to standard PCR.
• A strong negative correlation exists between annealing temperature and even primer utilization.
• DePCR with degenerate primers yields more consistent microbial community structures than standard PCR.

Conclusions:

• The DePCR method offers a simple yet effective way to mitigate PCR bias in complex microbial DNA amplification.
• It is particularly recommended for applications using degenerate primers or when primer-template mismatches are a concern.
• This protocol improves the accuracy of microbial community structure analysis, enhancing throughput and reliability.