Temperature-tolerant COLD-PCR reduces temperature stringency and enables robust mutation enrichment

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Low-level mutations in cancer samples are often missed, leading to diagnostic and management challenges.
• Coamplification at lower denaturation temperature PCR (COLD-PCR) enhances mutation detection but requires precise temperature control for single sequences.
• Existing COLD-PCR methods are limited to amplifying sequences with similar melting temperatures.

Purpose of the Study:

• To develop a temperature-tolerant (TT) approach to COLD-PCR (TT-COLD-PCR) that overcomes the limitations of precise temperature control.
• To enable simultaneous mutation enrichment in DNA amplicons with diverse melting temperatures.
• To increase the versatility and applicability of COLD-PCR for mutation detection.

Main Methods:

• Utilized thermocycling programs with gradually increasing denaturation temperatures.
• Enabled mutation enrichment by allowing cycling to reach the critical denaturation temperature of each amplicon.
• Validated TT-COLD-PCR using KRAS and TP53 exons, diluted mutated DNA, clinical samples, and circulating plasma DNA.

Main Results:

• A single TT-COLD-PCR program enriched mutations in amplicons with different melting temperatures simultaneously within a 2.5-3.0 °C window.
• Achieved 6-9-fold mutation enrichment with TT-full-COLD-PCR.
• Observed higher mutation enrichment with fast-COLD-PCR and ice-COLD-PCR variants.

Conclusions:

• TT-COLD-PCR allows simultaneous mutation enrichment in diverse amplicons with varying melting temperatures.
• This method enhances the detection of low-level mutations, improving diagnostic capabilities.
• TT-COLD-PCR significantly expands the utility of COLD-PCR technology in molecular diagnostics.